6H-Furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one, 2,3,3a,9a-tetrahydro-3-hydroxy-2-(hydroxymethyl)-7-methyl-, (2S,3S,3aR,9aS)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar - Nov 2010

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

(2S,3S,3aR,9aS)-3-hydroxy-2-(hydroxymethyl)-7-methyl-2,3,3a,9a-tertryhydro-6H-furo[2’,3’:4,5][1,3]oxazolo [3,2-a]pyrimidin-6-one

EC Number:

700-330-1

Cas Number:

433733-92-7

Molecular formula:

C10H12N2O5

IUPAC Name:

(2S,3S,3aR,9aS)-3-hydroxy-2-(hydroxymethyl)-7-methyl-2,3,3a,9a-tertryhydro-6H-furo[2’,3’:4,5][1,3]oxazolo [3,2-a]pyrimidin-6-one

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Anhydrothymidine
- Substance type: White powder
- Physical state: powder
- Analytical purity: 99.7%
- Purity test date: not stated
- Lot/batch No.: 073904/05
- Expiration date of the lot/batch: 11 December 2008
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar WI rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant.

Sex:

male

Details on test animals or test system and environmental conditions:

Wistar WI rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (6 weeks old at the start of treatment). The total number of animals used in the dose range finding study was 6 and in the main study 20. In the micronucleus main study 5 male rats were treated per sampling time in each treatment group.
The body weights of the rats at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 171.1 ± 10.1 g and the range 153 - 193 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatisation period was at least 6 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Animal husbandry
Room number
The animals were housed in room number 11 (dose range finding study) and 2 (main study).
Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 19.6 - 20.9°C), a relative humidity of 40 - 70% (actual range: 38 - 59%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the light/dark cycle (with a maximum of 4 hours) and the minimum level for humidity (with max 2%) occurred. Based on laboratory
historical data these deviations are considered not to affect the study integrity.
Accommodation
The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type MIV height: 18 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). Certificates of analysis of bedding were examined and then retained in the NOTOX archives.
Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline
The specific gravity of physiological saline is 1.0 g/ml.

Details on exposure:

The rats received an intraperitoneal injection of a maximum required dose of Anhydrothymidine. The route of administration was chosen to maximize the chance of the test substance reaching the target tissue.
The dosing volume was 10 ml/kg body weight. Anhydrothymidine concentrations were prepared on the day of administration.

Duration of treatment / exposure:

Dose range finding study: One dose group comprising of 3 males and 3 females received a single dose of Anhydrothymidine. This group was dosed with the highest concentration that was used for the main study. The observation period after each dosing was three days. During this period mortality and physical condition were recorded at least once a day.
Based on the results of the dose range finding test a limit test with one sex was performed. The test substance showed no toxicity in the dose range finding study up to 2000 mg/kg body weight, the highest dose required in the guidelines. Therefore, 2000 mg/kg was selected as the highest dose.
One dose level was used at both sampling times. The first sampling time was 24 h after treatment and the second sampling time was 48 h after treatment.
Five male rats were used per sampling time in each treatment group. The animals were dosed once.

Frequency of treatment:

a single dose

Post exposure period:

Bone marrow of the groups treated with Anhydrothymidine was sampled 24 or 48 hours after
dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone
marrow of the positive control group was isolated 48 hours after dosing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five male rats were used per sampling time in each treatment group.
In the dose range finding test, three male and three female animals dosed intraperitoneally with 2000 mg Anhydrothymidine per kilogram body weight.

Control animals:

yes

Positive control(s):

Cyclophosphamide, 30 mg/kg body weight

Examinations

Tissues and cell types examined:

Bone marrow of the groups treated with Anhydrothymidine was sampled 24 or 48 hours after
dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone
marrow of the positive control group was isolated 48 hours after dosing. The animals were
sacrificed using 02'C02• Both femurs were removed and freed of blood and muscles. Both ends
of the bone were shortened until a small opening to the marrow canal became visible. The bone
was flushed with approximately 4 ml of fetal calf serum (lnvitrogen Corporation, Breda, The
Netherlands). The cell suspension was collected and centrifuged at 1000 rpm (216 g) for 5 min.

Details of tissue and slide preparation:

The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet.
The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell
suspension was placed on the end of a clean slide, which was previously immersed in a 1: 1
mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a
tissue. The slides were marked with the NOTOX study identification number and the animal
number. The drop was spread by moving a clean slide with round-whetted sides at an angle of
approximately 45° over the slide with the drop of bone marrow suspension. The preparations
were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides
were prepared per animal.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals
should be above the historical control data range.
b) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test,
one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should
reasonably be within the laboratory historical control data range

Statistics:

Equivocal results should be clarified by further testing using modification of experimental
conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, onesided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at
any dose or at any sampling time) and the number of micronucleated polychromatic
erythrocytes in the animals was above the historical control data range.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant
(Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated
polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in
the animals was within the historical control data range.
The preceding criteria are not absolute and other modifying factors may enter into the final
evaluation decision.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that Anhydrothymidine is not clastogenic or aneugenic in the micronucleus test under the experimental conditions described in this report.