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:
20 August 2013 to 16 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: extremely viscous amber liquid
- Analytical purity:
- Lot/batch No.: E00031-68-1
- Expiration date of the lot/batch: 16 October 2010
- Storage condition of test material:room temperature in the dark, under nitrogen

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-633
- Retest date of the lot/batch: 30 January 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rodent bone marrow micronucleus test is currently the most commonly performed assay for detecting the genotoxic potential of test items in vivo. The original protocol for bone marrow micronucleus tests using small laboratory rodents was developed independently in the laboratories of Schmid and Heddle over 30 years ago. Since then, the methods and principles of the assay have remained largely unchanged. Most discussions on study design have therefore focused on the effects that different dosing and sampling schedules have on the micronucleus yields (Mavournin et al, 1990; Hayashi et al, 1994; Gatehouse, 1994; Trzos, 1978). The need for using both sexes for testing has also been a point of debate (Parton et al, 1990; Kliesch and Adler, 1992).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kent, England, UK.
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: Male: 201.7 - 239.4 g; Female: 174.3 - 201.2 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: All rats were housed in cages according to Charles River SOPs. Sterilised white wood shavings provided the bedding in the cages. Cages were changed on a weekly basis.
- Diet (e.g. ad libitum): Special Diet Services, Rat and Mouse (Modified) No. 1 SQC Expanded, was feely available.
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 - 21.4 °C
- Humidity (%): 50 - 73 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: Micronucleus test: From: 22 October 2013 To: 24 October 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: peanut oil
- Justification for choice of solvent/vehicle: Vehicle control
- Amount of vehicle (if gavage or dermal): 10 mL/kg/day
Details on exposure:
The oral route was selected as this is the a possible route of administration in humans. Based on dose range finder, the maximum recommended dose level of 2000 mg/kg/day was selected for the micronucleus test. As there were no clinical signs in the dose range finding test this was 2000 mg/kg/day only dose level tested.
All treated animals were exposed to test or control materials via the oral dose route. The rats were weighed immediately before each exposure event and the dose volume adjusted using a graded syringe fitted with a gavage needle. Treated animals were exposed to dosing formulations at a dose volume of 10 mL/kg/day.
Duration of treatment / exposure:
24 hours - the rats were treated at 0 h and 24 hours via the oral route at a dose level of 2000 mg/kg/day. Bone marrow samples were taken 24 hours after the final dose.
Frequency of treatment:
0 and 24 hours
Post exposure period:
24 hours
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Vehicle control: 5 males and 5 females
EXP0700332: 7 males and 7 females
Positive control: 3 males
Untreated control: 3 males and 3 females
Control animals:
yes
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): An adequate positive control response for at least 2 animals and the dose group as a whole. This criterion was met in this micronucleus test.
- Route of administration: oral
- Doses / concentrations: 10 mL/kg/bw (dose 50 mg/kg; 5 mg/mL)

Examinations

Tissues and cell types examined:
The frequenct of reticulocytes and micronucleated cells were determined. The number of micronucleated normochomatic erythrocytes in mature red blood corpulscles were also recorded as a control.
Scored micronuclei were also assigned based on size which provided a non-specific measure of compound induced spindle dsfunction
Details of tissue and slide preparation:
Rats were killed by increasing levels of carbon dioxide. The marrow was flushed out using a 1:1 mixture of foetal calf serum and trisodium citrate (0.8 %, w/v) in Sorenson's buffer (pH 6.8). Routine tissue culture antibiotics were included to prevent microbial growth. This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. Following completion of the sampling procedure the contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells.
The tubes were centrifuged to pellet the cells. The supernatant fluid was discarded and the cells were then resuspended on a vortex mixer in the residual liquid.
Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube (animal). The smear was left to air dry, fixed in methanol (for at least 5 min) and then stained with Giemsa stain (15%, v/v, in water) to give optimum erythrocyte discrimination. Permanent slide preparations were made by sealing cover slips onto the glass slides using mounting medium.
Evaluation criteria:
The average micronucleus incidence in vehicle control dosed and untreated CD rats, has in this laboratory been determined as 0.04% , a range of 0.01 – 0.13% per group of 5-7 animals and 0.02 - 0.11% per group of 10-12 animals.
These historical data have been used in the evaluation of response in this test. This frequency is also in agreement with published data for micronucleus tests with rats (Tamura et al, 1990; Salamone and Mavournin, 1994).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No micronucleus induction was detected in bone marrow erythrocytes of rats dosed with EXP0700332.

Applicant's summary and conclusion

Conclusions:
In conclusion, EXP0700332 did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female Sprague Dawley (CD) rats using a 0 h and 24 h oral dosing and 48 h sampling regimen.
Executive summary:

The in vivo genotoxic potential of EXP0700332 was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female rats following a 0 h and 24 h oral dosing and 48 h sampling regimen.

A Dose Range Finding study was undertaken to establish a suitable dose range for the main micronucleus experiment. Based on the findings of the Dose Range Finder, the maximum recommended dose of 2000 mg/kg/day was judged to be a suitable dose level for the micronucleus test.

In the micronucleus test, groups of rats were dosed at 0 h and 24 h via the oral route with EXP0700332 at the maximum recommended dose level, 2000 mg/kg/day. Bone marrow samples were taken 24 h after the final dose. Two control groups of rats were also dosed orally with either the vehicle, peanut oil (10 mL/kg/day), or the positive control agent, cyclophosphamide (50 mg/kg/day). An untreated control group was also included. The experimental schedule for the control groups followed that of the test item treated rats.

No micronucleus induction was detected in bone marrow erythrocytes of rats dosed with EXP0700332.

Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

In conclusion, EXP0700332 did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female Sprague Dawley (CD) rats using a 0 h and 24 h oral dosing and 48 h sampling regimen.