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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:
Between 2007-08-22 and 2007-09-21.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP study with no deviations.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
-
EC Number:
451-530-8
EC Name:
-
Cas Number:
736150-63-3
Molecular formula:
Not applicable
IUPAC Name:
Not allocated
Details on test material:
- Name of test material (as cited in study report): TS-ED 532
- Lot/batch No.: item 175540 batch 4010534806
- Expiration date of the lot/batch: 31 December 2009
- Stability under test conditions: stable
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: albino Crl:CD-1™(ICR)BR strain mice supplied by Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately five to eight weeks old.
- Weight at study initiation: 23 to 30g.
- Assigned to test groups randomly: randomly
- Fasting period before study: No information
- Housing: the animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Diet (e.g. ad libitum): free access to food (Certified Rat and Mouse Diet Code 5LF2, IPS Ltd., London, UK).
- Water (e.g. ad libitum): free access to mains drinking water.
- Acclimation period: seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness.

IN-LIFE DATES: From: 22 August 2007 To: 21 September 2007

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis oil
- Justification for choice of solvent/vehicle: Arachis oil usually used by the testing lab for this type of study.
- Concentration of test material in vehicle: 100 and 200 mg/ml.
- Lot/batch no. (supplier): P72
- Lot/batch no. (testing lab): V-4046
- Date received : 20 April 2007
- Description : Pale straw coloured liquid
- Storage conditions : Room temperature
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): TS-ED 532 was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.



Duration of treatment / exposure:
Range-finding test: 48 hours
Main test: 24 or 48 hours
Frequency of treatment:
Range-finding test: A single dose via oral by gavage or intraperitoneal routes
Main test: A single dose via intraperitoneal route
Post exposure period:
NA
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 and 2000 mg/kg bw in the range-finding test and 2000 mg/kg bw in the main test
Basis:
nominal conc.
No. of animals per sex per dose:
Range-finding test:
1 male/1 female (1000 mg/kg bw via oral by gavage)
1 male/1 female (2000 mg/kg bw via oral by gavage)
2 male/2 female (2000 mg/kg bw via intraperitoneal route)
2 male (2000 mg/kg bw via intraperitoneal route).

Main test:
7 male (arachis oil via intraperitoneal route) - Vehicle control group (24 hours time point)
7 male (arachis oil via intraperitoneal route) - Vehicle control group (48 hours time point)
5 male (cyclophosphamide via intraperitoneal route) - Positive control group (24 hours time point)
7 male (TS-ED 532 via intraperitoneal route) - Test group (24 hours time point)
7 male (TS-ED 532 via intraperitoneal route) - Test group (48 hours time point)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide is a positive control material known to produce micronuclei in this type of study
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Erythrocytes from bone marrow tissue.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A limit test at the maximum recommended dose of 2000 mg/kg bw was performed based upon the results from the range-finding test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Immediately following termination (i.e. 24 or 48 hours following dosing), the femurs from all animals were dissected and aspirated with foetal calf
serum. The bone marrow was extracted and smears prepared following centrifugation and re-suspension of bone marrow tissue.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were air-dried, fixed in absolute methanol, stained in May-Grüwald/Giemsa staining solution, allowed to air-dry and
cover-slipped using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blindly using light micropsy at x1000 magnification. The incidence of micronucleated cells
per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic
erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted and further scored for incidence of mcronuclei.
The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio) was calculated together with appropriate group mean values and
standard deviation.
Evaluation criteria:
A statistically significant dose-responsive and toxicologically relevant increase in the number of micronucleated PCE erythrocytes in
comparison with corresponding control group, indicates a positive mutagenic response.

A statistically significant lower PCE/NCE ratio when compared to corresponding control group indicates a positive response for bone marrow
toxicity.
Statistics:
The data was analysed following a square root (x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Range-finding test:
No evidence of toxicity (premature death or clinical signs) was observed in animals dosed with TS-ED 532 via the oral and intraperitoneal route
at dose levels up to the maximum recommended dose (2000 mg/kg bw).
No marked difference was observed between male and female mice.

Main test:
Based on the results of the range-finding test a limit test at 2000 mg/kg bw was performed in male mice. The intraperiotoneal route of administration
was used to ensure maximal systemic exposure.

There were no differences in the frequency of micronucleated PCEs or in the PCE/NCE ratios for the TS-ED 532 test groups (24 or 48 hours), when
compared to their concurrent control groups.

The positive control group induced a statistically significant increase in the number of micronucleated PCEs, hence confirming the the sensitivity of the test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
TS-ED 532 was considered to be non-genotoxic in vivo in the micronucleus test. It did not induce damage to chromosomes or aneuploidy.
Executive summary:

TS-ED 532 was tested for the potential to induce damage to chromosomes or aneuploidy when administered to mice in the micronucleus test (OECD 474). Initially, a range-finding test was performed to establish dose levels, route of administrations and differences in toxic responses between the sexes.

The main test was conducted as a limit test using groups of seven male mice at the maximum recommended dose (2000 mg/kg bw). The intraperitoneal route was used to maximise systemic TS-ED 532 exposure. The animals were killed 24 or 48 hours later, the bone marrows were extracted, and smears were prepared and stained. Polychromatic (PCE) and normachromatic erythrocytes (NCE) were scored for the presence of micronuclei. Two vehicle groups of 7 mice each were given a single intraperitoneal dose of arachis oil and killed 24 or 48 hours later. One positive control group of 5 mice were given a single oral dose of cyclophosphamide and killed after 24 hours.

TS-ED 532 did not induce a statistically significant decrease in the PCE/NCE ratio in the 24 or 48 hours test groups when compared to their concurrent control groups. TS-ED 532 did not induce a statistically significant increase in the frequency of micronucleated PCEs when compared to their concurrent control groups. The positive control group induced a marked increase of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the tests system.

TS-ED 532 was found to be non-genotoxic in mice when tested in the micronucleus test.