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)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

It is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and it is not clastogenic in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May - 05 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 97, TA 98, TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA100
First assay: 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA1535, TA97, TA98 and TA102
Second assay: 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA1535, TA97, TA98, TA100 and TA102

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 5 μg/plate in saline for TA1535

Positive controls:

yes

Positive control substance:

other: ICR-191 1 μg/plate in DMSO for TA97

Remarks:

Without metabolic activation (-S9-mix)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 10 μg/plate in DMSO for TA98

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 650 μg/plate in DMSO for TA100

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 0.1 µg in DMSO for TA102

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO (5 and/or 10%) for all tester strains

Remarks:

With metabolic activation (-S9-mix) for all tested strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.


DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies


OTHER EXAMINATIONS:
- Determination of precipitation

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose range finding test with tester strain
TA100 with and without S9-mix. Eight concentrations, 3, 10, 33,100,333, 1000, 3330 and
5000 μg/plate were tested in triplicate. This dose range finding test was reported as a part of the
first experiment of the mutation assay. The highest concentration of Anhydrothymidine used in
the subsequent mutation assay was 5 mg/plate.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 97, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: no increase in the number of revertants was observed upon treatment with Anhydrothymidine under all conditions tested.


COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Precipitate

Precipitation of Anhydrothymidine on the plates was not observed at the start or at the end of the incubation period.

Toxicity

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity

In both mutation assays, no increase in the number of revertants was observed upon treatment with Anhydrothymidine under all conditions tested.

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
It is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May - 2 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 mg/ml respectively) and 30 U/ml heparin
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

First assay: 333, 1000 and 2402 mg/ml culture medium with and without S9 mix (3 h exposure time, 24 h fixation time)
Second assay: 333, 1000 and 2402 mg/ml culture medium without S9 mix (24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time)
Second assay: 333, 1000 and 2402 mg/ml culture medium with S9 mix (3 h exposure time with a 48 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s): dimethyl sulfoxide

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: in Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix) Migrated to IUCLID6: in Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: 48h
- Exposure duration:
First experiment: 3 h in the absence and presence of S9-mix,
Second experiment: 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells):
First experiment: 24 h
Second experiment: 24 h and 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in tap water.


NUMBER OF REPLICATIONS: duplicates in two independent experiments


NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (was determined by counting the number of metaphases per 1000 cells of each culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Anhydrothymidine was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Anhydrothymidine for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
The highest tested concentration was 2402 μg/ml (= 0.01 M).
After 3 h exposure to Anhydrothymidine in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix
were not rinsed after exposure but were fixed immediately (24 hand 48 h fixation time).
Cytotoxicity of Anhydrothymidine in the lymphocyte cultures was determined using the mitotic index.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Anhydrothymidine did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Anhydrothymidine on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Anhydrothymidine does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

It is concluded that this test is valid and that the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

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.

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.

Dose / conc.:

0 mg/kg bw/day (nominal)

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

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The product showed negative results in in vitro (AMES test and Chromosome Aberration Study) and in vivo (Micronucleus Test) genotoxic studies.