2,4(1H,3H)-Pyrimidinedione, 1-(3,5-di-O-acetyl-2-deoxy-.beta.-L-erythro-pentofuranosyl)-5-methyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of this study it is concluded that LDT600 C3 is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Diacetyl Thymidine (LDT600-C3) is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

QSAR- in vitro gene mutation study in mammalian cells: negative

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:

23.5.2009 - 29.5.2009

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

other: TA97, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

100, 333, 1000, 3330 and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide (TA1535), 2-nitrofluorene (TA98), methylmethanesulfonat (TA100), cumene hydroperoxide (TA102), ICR-191 (TA97); with S9: 1,8 dihydroxyanthraquinone (TA102), 2-aminoanthracene (all other strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: three replications in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn

Evaluation criteria:

A test substance is considered positive in the test, if:
- The total number of revertants in tester strains TA100, TA97 and TA102 is greater than two times the concurrent control or the total number of revertants in tester strains TA1535 and TA98 is greater than three times the concurrent control.
- In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

other: TA97, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The test substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains (TA1535, TA97, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

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 the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that LDT600 C3 is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2019 - Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
The highest tested concentration was 2000 μg/mL exposure medium.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity.
After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale Recommended test system in international guidelines (e.g. OECD).
Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 55 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.1 – 37.4°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
Stock cultures of the cells were stored in the freezer (-150°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/mL.
Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control
was tested in duplicate. In the first experiment, cell cultures were exposed for 3 hours to Diacetyl Thymidine (LDT600-C3) in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to Diacetyl Thymidine (LDT600-C3) in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium
(R10-medium). The cells in the final suspension were counted with the coulter particle counter.

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

In addition to the criteria stated below, any increase of the mutation frequency should be
evaluated for its biological relevance including comparison of the results with the historical
control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the
negative/solvent MF distribution plus one standard deviation. For the micro well version of
the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of
more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be
biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion
for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested
concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Diacetyl Thymidine (LDT600-C3) is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

January 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

The prediction was considered satisfactory because of the structural similarity and similar mechanism of action of the target chemical and the analogs.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The chemical was entered in the QSAR Toolbox by SMILES. The identified structure of the chemical was
retained and subsequently added to the data matrix. After selecting the endpoint of interest
(genotoxicity), in the data module, the relevant databases were searched for experimental data. No
experimental data was found. The profiling module was used to identify the main characteristics of the
chemical. In category definition, the chemical profiler (OECD HPV chemical categories) was used to
categorize the chemical. In the data filling, in vitro genotoxicity test results with L5178Y mouse
lymphoma cells was selected because there was sufficient analog data available to be used to fill the
data gap. The data gap filling was initiated by read-across, as it is the appropriate data gap-filling
method for qualitative endpoints such as genotoxicity. The results were subsequently refined by DNA
binding by OECD, protein binding alerts for chromosomal aberration by OASIS, DNA alerts for CA and
MNT by OASIS and organic functional groups (US EPA). The prediction was considered satisfactory
because of the structural similarity and similar mechanism of action of the target chemical and the
analogs.

GLP compliance:

not specified

Target gene:

QSAR

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

not measured/tested

Database(s) used:

- ECHA CHEM

Category boundaries (applicability domain):

- Active descriptor(s) range:

- log Kow: from -0.38 to 0.23 target chemical is in domain

- Response range:

- in vitro gene mutation study in mammalian cells: negative (x9) (from Negative to Negative)

Profilers:

- Tertiary Amines (OECD HPV Chemical Categories) (primary grouping)

target chemical is in domain

- DNA binding by OECD (subcategorization)

target chemical is in domain

- Protein binding alerts for Chromosomal aberration by OASIS (subcategorization)

target chemical is in domain

- DNA alerts for CA and MNT by OASIS (subcategorization)

target chemical is in domain

- Organic functional groups (US EPA) (subcategorization)

target chemical is in domain

Conclusions:

in vitro gene mutation study in mammalian cells: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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 the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that LDT600 C3 is not mutagenic in the Salmonella typhimurium reverse mutation assay.

In the absence of S9-mix, Diacetyl Thymidine (LDT600-C3) did not induce a biologically

relevant increase in the mutation frequency in the first experiment. This result was confirmed

in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, Diacetyl Thymidine (LDT600-C3) did not induce a biologically

relevant increase in the mutation frequency.