Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2005 to February 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed by OECD guidelines and under GLP with no deviations. All finding clearly support the "not clastogenic" conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report: MTDID-5789
- Substance type: colorless liquid
- Physical state: volatile liquid
- Analytical purity: 99.91%
- Lot/batch no.: Lot #2
- Expiration date of the lot/batch: 31 October 2007
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7-22.1 deg C.
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: October to Dec 2005

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: [none; no data; acetone; air; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil ; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; water] 1% CMC
- Justification for choice of solvent/vehicle: Suitably stable emulsion
- Concentration of test material in vehicle:200 mg/mL
- Amount of vehicle (if gavage or dermal): 10mL/kg
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
once
Post exposure period:
24, 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
other: nominal injection
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; 2-acetylaminofluorene; 2-nitrofluorene; 3-methylcholanthrene; 4-nitroquinoline-N-oxide; 7,12-dimethylbenzanthracene; 9,10-dimethylbenzanthracene; 9-aminoacridine; benzo(a)pyrene; congo red; cumene hydroperoxide; cyclohexylamine; cyclophosphamide; ethylmethanesulphonate; ethylmethanesulphonate; ethylnitrosurea; ethylnitrosurea; furylfuramide; ICR 191; methylmethanesulfonate; mitomycin C; mitomycin C; monomeric acrylamide; N-dimethylnitrosamine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-nitrofluorene; 4-nitroquinoline 1-oxide; sodium azide; triethylenemelamine

cyclophosphamide;

- Justification for choice of positive control(s): Common usage
- Route of administration:
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: According to OECD Guidelines
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single injections with collection after 24 and 48 hours.
DETAILS OF SLIDE PREPARATION:
Slides were automatically stained using the "Wright-stain-procedure"

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. Averages and standard deviations were calculated
Evaluation criteria:
All validation critieria were met.
a. Positive control substance performed as desired.
b. Incidence of micronucleated polychromatic erhthrocytes were within historical control ranges. Mean +/- 3 standard deviations

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Solubility: suspension
- Clinical signs of toxicity in test animals: negative
- Evidence of cytotoxicity in tissue analyzed:none
- Rationale for exposure: well tolerated high dose
- Harvest times: Study duration of 3 days

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): None
- Ratio of PCE/NCE (for Micronucleus assay): None
- Appropriateness of dose levels and route: Appropriate
- Statistical evaluation: Yes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Not clastogenic under the conditio0ns of this assay
Executive summary:

MTDID 5789 (Lot #2) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. Batch Lot #2 of MTDID 5789 was a clear colourless liquid with a purity of 99.91%. The test substance was emulsified in 1% (w/v) carboxymethylcellulose. Five male animals were used in each of the four treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with MTDID 5789 at 2000 (two groups) mg/kg body weight. All animals showed no abnormalities after dosing. Bone marrow of the groups treated with MTDID 5789 was sampled 24 or 48 hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with MTDID 5789. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with MTDID 5789 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that MTDID 5789 is not clastogenic in the micronucleus test under the experimental conditions described in this report.