Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2013 to 07 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
other: The mammalian in vivo micronucleus test detects damage induced by the test chemical to the chromosomes or the mitotic apparatus of erythroblasts. It evaluates micronucleus formation in erythrocytes sampled from bone marrow or peripheral blood cells.

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Test Substance I.D.: EXP1200078
Test Substance Lot Number: E00275-350
Test Substance Purity: 100%
Test Substance Description: Blackish orange clear viscous liquid

Test animals

Species:
rat
Strain:
other: Sprague Dawley Hsd:SD rats
Details on species / strain selection:
6 weeks old at study initiation
Sex:
male
Details on test animals and environmental conditions:
Housing
Animals were housed in a controlled environment at 72 ± 3¿F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips were used for bedding to absorb liquids. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Housing
Animals were housed in a controlled environment at 72 ± 3¿F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips were used for bedding to absorb liquids. A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle control was Mineral Oil (CAS No.: 8042-47-5, Lot No.: MKBP0802V, Expiration Date: 31 March 2018 obtained from Sigma Aldrich. The dose formulation was prepared prior to dose administration.
Details on exposure:
A single oral dose at 2000 mg/kg was given via gavage. The dose was based on results of the 28d and 90d repeat dose studies which demonstrated that 2000 mg/kg was the MTD and well tolerated.
Duration of treatment / exposure:
The were two groups of control/vehicle and two groups at the MTD (2000 mg/kg). Group 1 was kept for 24 hours, and group 2 was kept for 48 hours.
Frequency of treatment:
One single dose.

Dose Administration and Observation
All dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay. Body weights were recorded prior to the first dose for the purpose of dose volume calculations. Animals were observed prior to dose, approximately one and two hours after dose administration and daily thereafter for clinical signs of toxicity.
Post exposure period:
Group 1 - 24 hours
Group 2 - 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
MTD based on OECD 407 and OECD 409
No. of animals per sex per dose:
Group 1 - 5 animals
Group 2 - 5 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide; 40 mg/kg; duration = 24 hours.

Examinations

Tissues and cell types examined:
Femoral bone marrow was collected at approximately 24 or 48 hours after the single dose.
Details of tissue and slide preparation:
Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Evaluation criteria:
Bone marrow was evaluated by fluorescent microscopy. The staining procedure permitted the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost like, dark green NCEs, respectively). The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei.

At least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE/EC proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data was excluded from evaluation. The frequency of mnPCEs and the proportion of PCEs to total erythrocytes was determined for each animal and treatment group.
Statistics:
Statistical significance (p = 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Criteria for a Valid Test
The vehicle control group should be consistent with the historical vehicle control range, and must be = 0.4% mnPCEs (Aikihiro et al., 1998), and the positive control must induce significant increase (p = 0.05) in mnPCE frequency as compared to the concurrent vehicle control.

Five animals/group were available for analysis

Any other information on results incl. tables

Evaluation of Test Results

Once the criteria for a valid assay were met, the results were evaluated. Test substance was considered to be positive if it induced a significant increase in mnPCE frequency (p£0.05) at any dose level or sampling time compared to the concurrent vehicle control. The test substance was considered to be negative if no significant increase in mnPCE frequency was observed (p > 0.05) compared to the concurrent vehicle control. Other criteria may have been used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Definitive Micronucleus Assay

No mortality occurred during the course of the definitive assay. All rats appeared normal throughout the observation period. Clinical signs are presented inTable 1.

Bone Marrow Analysis

No appreciable reductions in the PCEs/EC ratio in the test substance group compared to the vehicle control group were observed indicating the test did not induce cytotoxicity.

No statistically significant increase in the incidence of mnPCEs in the test-substance treated group was observed relative to the negative control group (p > 0.05, Kastenbaum-Bowman tables). The positive control induced a statistically significant increase in the incidence of mnPCEs (p < 0.05, Kastenbaum-Bowman tables). The number of mnPCEs in the vehicle control groups did not exceed the historical control range.

The incidence of mnPCEs per 10,000 PCEs scored (2000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time inTable 2. Individual animal data is presented inTable 3. 

Based upon this, all criteria for a valid test were met as specified in the protocol. The Common Technical Document (CTD) Summary Table is included inAppendix IV.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the administration of EXP1200078 at a dose of 2000 mg/kg was concluded to be negative in the Micronucleus assay.
Executive summary:

A well conducted guideline study to examine in vivo mutagenesis (chromosome aberration) of EXP1200078 at the MTD (2000 mg/kg) did not result in any mutagenic response. The test substance, EXP1200078,was evaluated for its clastogenic activity and/or disruption
of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow.

No adverse effects were found at the dose of 2000 mg/kg in this in vivo genetic toxicity assay.