Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to guideline
Guideline:
other: ICH S2A (1995)
Deviations:
no
Remarks:
The study was conducted according to the guidelines in effect at the time of study conduct
GLP compliance:
yes
Type of assay:
other: Rat bone marrow chromosome aberration/micronucleus test

Test material

Constituent 1
Reference substance name:
Lecithins, acetylated
EC Number:
293-316-5
EC Name:
Lecithins, acetylated
Cas Number:
91053-50-8
Molecular formula:
Not applicable- complex UVCB substance
IUPAC Name:
Lecithins, acetylated
Details on test material:
Purity: Unknown variable composition biological substance (UVCBS)
Composition of test material, percentage of components: Unknown variable composition biological substance (UVCBS)

Test animals

Species:
rat
Strain:
other: Sprague-Dawley SD® (Hsd:SD®)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately59 days old
- Weight at main study start: Males weighed 228-275 g and females weighed 164-195 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Socially housed 2 or 3 animals of the same sex within a group in stainless steel perforated floor cages
- Diet (e.g. ad libitum): ad libitum except during designated procedures
- Water (e.g. ad libitum): ad libitum except during designated procedures
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle: Corn Oil NF
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared up to one day prior to dosing. The required amount of test substance was weighed using a positive displacement pipette and transferred to an appropriate 50 mL plastic tube. Approximately 20 mL of the vehicle was added and the formulation was vortexed until mixed. A measuring cylinder was pre-rinsed with the vehicle and the content of the plastic tube was transferred into the cylinder. The plastic tube was rinsed with a small amount of vehicle. The cylinder was inverted to mix then the contents sonicated 5-10 minutes to de-foam the suspension. The formulation was then brought to final volume with corn oil and inverted to mix. The dose formulations were maintained on stir plates throughout the dosing procedure.

The positive control solution was freshly prepared on the day of use.
Duration of treatment / exposure:
Animals were given a single oral dose via intragastric gavage.
Frequency of treatment:
Once
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:

No. of animals per sex per dose:
Vehicle: 5/sex (sampling time 18 hours); 5/sex (sampling time 42 hours)
500 mg/kg: 5/sex (sampling time 18 hours)
1000 mg/kg: 5/sex (sampling time 18 hours)
2000 mg/kg: 5/sex (sampling time 18 hours); 5/sex (sampling time 42 hours)

Positive control: 3/sex (sampling time 18 hours)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide was given via gavage at a dose of 20 mg/kg.

Examinations

Details of tissue and slide preparation:
Bone marrow prep for micronucleus test: After centrifugation, each resulting cell pellet was resuspended in 2 mL of filtered foetal bovine serum and re centrifuged. The cell pellet was resuspended in a small volume of foetal bovine serum to facilitate smearing in the conventional manner on glass microscope slides. Several smears were prepared from each animal and fixed in methanol for at least 10 minutes.

Bone marrow prep for chromosome aberration test: After centrifugation, each resulting cell pellet was resuspended in 10 mL aqueous 0.075M potassium chloride (hypotonic solution) and incubated for approximately 12 minutes at ca. 37°C, before addition of 2 mL of fixative (3 vol methanol:1 vol acetic acid) with mixing. Following centrifugation, the supernatant was discarded and the cells treated with 3 changes of neat fixative. After the third change of fixative, the cell pellet was collected by centrifugation and resuspended in fixative at an appropriate density for slide preparation. The fixed cells were dropped onto clean slides and air-dried before staining. At least two slides were prepared from each animal. Fixed cells not used for slide preparation were discarded after completion of the experimental phase of the study.

Slide staining for micronucleus test: The slides for examination were encoded to minimize potential operator bias and then mounted temporarily in aqueous Acridine Orange.

Slide staining for chromosome aberration test: The slides were washed with 3 changes of purified water (approximately 1 minute per wash) then stained with 10% (v/v) Giemsa for 15 minutes, rinsed in purified water then washed in running tap water for approximately 5 minutes, air-dried then mounted with coverslips using synthetic mountant.

Microscopic Examination - Micronucleus Test: The slides were randomized and encoded to minimize potential operator bias and then examined by fluorescence microscopy using a blue excitation filter and a yellow barrier filter. A total of 2000 immature erythrocytes per animal were examined for the presence of micronuclei. Usually only one smear was examined per animal, the remaining smears were held temporarily as reserves in case of technical problems with the first smear.

In addition, the proportion of immature erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal. The incidence of any micronucleated mature erythrocytes observed during this assessment was recorded as a check for potential micronucleus like artifacts.

Microscopic Examination - Chromosome Aberration Test:

Mitotic Index (MI) - Slides were randomized then encoded to minimize potential operator bias. They were examined by light microscopy, and the mitotic index was determined by examination of at least 1000 cells per animal. The relative mitotic index (RMI) was calculated as a percentage ratio compared with the concurrent vehicle control group.

Detailed Examination for Chromosome Aberrations - Slides were examined by light microscopy, and (where practical) a total of 100 readable metaphases per animal was examined for the presence of chromosome aberrations using oil immersion optics.
Evaluation criteria:
Micronucleus Test - A positive response is normally indicated by a statistically significant dose related (where appropriate) increase in the incidence of micronucleated immature erythrocytes. A negative result is indicated when there is no dose related increase in the incidence of micronucleated immature erythrocytes and when individual and group mean values fall within (or close to) the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Chromosome Aberration Test - Where statistical analysis is performed, a positive response is normally indicated by a statistically significant (dose related, if applicable) increase in the incidence of aberrant cells for the treatment group compared with the concurrent control group. A negative result is indicated where group mean incidences of aberrant metaphase cells for the group treated with the test substance are not significantly greater than incidences for the concurrent control group and where these values fall within or close to the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Statistics:
See additional information below for statistical analyses.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant clinical signs of reaction to treatment or mortalities were observed for any of the treatment groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: Negative
Animals treated with the test substance did not show any significant increases in the proportion of micronucleated immature erythrocytes or aberrant metaphases at either sampling time. It is therefore concluded that the test substance did not show any evidence of genotoxicity in the combined rat bone marrow chromosome aberration/micronucleus test, when tested in accordance with regulatory guidelines.

Executive summary:

The purpose of this study was to evaluate the genotoxicity of the test substance using the combined rat bone marrow chromosome aberration/micronucleus test. Young adult rats were treated with a single administration of the control article (corn oil), positive control (cyclophosphamide) or the test substance (500, 1000, or 2000 mg/kg), orally by intragastric gavage. No significant clinical signs of reaction to treatment or mortalities were observed for any of the groups.

 

Animals were euthanized 18 or 42 hours after treatment (3 hours after colchicine administration) and bone marrow from the femurs was collected. For the micronucleus test, bone marrow smears prepared (from the right femur) were fixed, temporarily stained with Acridine orange, and examined under code using fluorescence microscopy. A total of 2000 immature erythrocytes per animal were examined for the presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes was assessed for each animal as a measure of potential bone marrow toxicity. For the chromosome aberration test, bone marrow cells from the left femur were treated with hypotonic solution then fixed, dropped onto clean slides, air-dried, stained with Giemsa and examined under codeusing light microscopy. The mitotic index was determined by examination of at least 1000 cells per animal. The relative mitotic index was calculated as a percentage ratio compared with the concurrent vehicle control group. A total of 100 readable metaphases per animal were examined for the presence of structural and numerical chromosome aberrations.

 

Animals treated with the test substance did not show any statistically significant increases in the incidence of micronucleated immature erythrocytes or any significant decreases in the proportion of immature erythrocytes. In addition, the incidences of micronucleated immature erythrocytes all fell within the laboratory historical control range. Animals treated with the test substance did not show any statistically significant increases in the proportion of aberrant metaphases at any experimental point. In addition, the proportion of aberrant metaphases for vehicle and test article groups was within or close to the laboratory historical control range. The positive control agent caused substantial increases in the proportions of micronucleated immature erythrocytes and aberrant metaphases for each individual animal confirming the sensitivity of the test.

 

Animals treated with the test substance did not show any significant increases in the proportion of micronucleated immature erythrocytes or aberrant metaphases at either sampling time. It is therefore concluded that the test substance did not show any evidence of genotoxicity in the combined rat bone marrow chromosome aberration/micronucleus test, when tested in accordance with regulatory guidelines.