2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 10993-5:2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USP 35 chapter <87>

Deviations:

not specified

Principles of method if other than guideline:

Following the recommendations in Annex D of ISO 1099-5, the quatification of the test for in vitro cytotoxicity was performed using the XTT method, based on the metabolism of yellow XTT to a red formazan.

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: cytotoxicity

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 103901

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 20 ± 5 °C
- Stability under test conditions: stable
- Solubility in water: 7mM

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolution in medium

Species:

mouse

Strain:

other: Cell line L-292 (DSMZ no: ACC 2)

Details on test animals or test system and environmental conditions:

CELL LINE:
-The study was performed on cell line L-929 (DSMZ no.: ACC 2), of murine (mouse) (Mus musculus). This is a connective tissue fibroblast, established from the normal subcutaneous areolar and adipose tissue of a male C3H/An mouse.
-Source: RD Biotech
-Batch: 110425-JFM

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37 ± 1 °C
- CO2: 5 ± 1.5%

Route of administration:

other: Direct exposure of sub-cultured to dosed medium

Vehicle:

other: Dulbecco's modified eagle medium with fetal calf serum at 10%

Details on exposure:

The test item was prepared 24 ± 2h before starting the exposure. Three replicates were used for test samples and controls. The test was performed in flat-bottom 96-well micro-titer plates using approx. 1000 viable cells per well.
The study was performed on the original soultion and a dilution series of the solution using the vehicle as a diluent with the final concentrations of the test item n the cell cultures of 7 mM, 1.4mM, 0.28 mM and 0.056 mM.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

24 ± 2h

Frequency of treatment:

Single exposure

Post exposure period:

No post-exposure period

Dose / conc.:

7 other: mM

Dose / conc.:

1.4 other: mM

Dose / conc.:

0.28 other: mM

Dose / conc.:

0.056 other: mM

No. of animals per sex per dose:

N.a.

Control animals:

other: In vitro test: not animals, but cell cultures exposed to positive and negative control

Details on study design:

As a positive control, the Positive Bioreaction 10cmx1cm USP Reference Standard (USP1071439) was used. As a negative control, the Polyethylene High Density reference 1546707 was used as described in the CONFARMA SOP BC 5 as replacement of the test item in the control extraction. Both positive and negative controls were extracted for 72 ± 2h at 37 ± 1°C under agitation. The positive controls were analysed in parallel to the test item using the same cells and the same reagent preparations. To check for systematic cell seeding errors, Growth Controls were placed both at the left side (row 2) and the right side (row 11) of the 96-well plate (row 1 and row 12 were not used for cells). Blanks were placed in row 1 and 12 to ascertain correct disctribution of the XTT reagent.

A decrease in number of living cells results in a decrease in the metabolic activity in the sample. This decrease directly correlates to the amount of formazan formed, as monitored by the optical density at 480 nm. If viability is reduced to <70%, the test item is considered to have cytotoxic potential.

Acceptance criteria:
-The left and right mea of the blanks do not differ by more than 15% form the mean of all blanks.
-The left and the right mea of the Growth Controls do not differ by more than 15% form the mean of all Growth Controls.
-The 50% solution of the test sample should have at least the same or a higher viability than the 100% solution, taking into account the variation of the individual results.
-The replicates for the test item dilutions show a variation of ≤20%.
-The growth control and the egative control show a grading of "0".
-The positive control has a cytotoxic effect.

Examinations:

During the test, cell cultures were routinely tested to be free of Mycoplasma using PCR and regularly checked to see changes in their doubling time and morphology.

After exposure, the condition of the cells was evaluated using a phase-contrast microsope. The morphonlogy and density of the cells was graded. The formazan formation was determined for each treatment concentration and compared to that determined in control cultures.

Positive control:

As a positive control, the Positive Bioreaction 10cmx1cm USP Reference Standard (USP1071439) was used. The positive control showed a cytotoxic effect on the L-929 cells, with a reduction of viaility to less than 70% for the dilutions of 100 to 8.8% (v/v) and a grading of "4" for all dilutions.

Details on results:

The viability in the quantitative assay was at least 84.1% or higher for cells incubated with the test item compared to the negative control. The grading was "0" for all dilutions tested. The test for cytotoxicity fulfilled all acceptance criteria. The reuslts obtained for the test item can therefore be considered to be valid.

Conclusions:

No cytotoxic effect was observed in L-929 cells upon exposure to the test item.

Executive summary:

The study was designed to determine the in vitro cytotoxicity of SP02 in a cell culture assay, using a solution of the test item prepared in cell culture medium with serum. The test item was used for the study in 4 concentrations, 7mM, 1.4 mM, 0.28mM and 0.056 mM, respectively.

The suitability of the test item was confirmed by the reaction of the cells to the negative and positive controls. Incubation of the cells with the control medium and the negative control (Polyethylene High Density reference 1546707) resulted in viabilities of at least 96.5% and 105.0%, respectively and a gradig of "0" for all negative controls. The positive control (USP1071439) was proven to be cytotoxic in the dilutions 100 to 8.8% V/V. The assay fulfilled all acceptance criteria.

The quantification of the viability of the cells after 24 ± 2 h incubation with the test item showed a minimal viability of 84.1% in the dilution of 7 mM and a grading of "0" for all concentrations tested. In conequence, no cytotoxic effect was demonstrated for SP02.

Description of key information

The cytotixcicity study was performed determine the in vitro cytotoxicity of SP02 in a cell culture assay, using a solution of the test item prepared in cell culture medium with serum. The test item was used for the study in 4 concentrations, 7mM, 1.4 mM, 0.28mM and 0.056 mM, respectively.

The suitability of the test item was confirmed by the reaction of the cells to the negative and positive controls. Incubation of the cells with the control medium and the negative control (Polyethylene High Density reference 1546707) resulted in viabilities of at least 96.5% and 105.0%, respectively and a gradig of "0" for all negative controls. The positive control (USP1071439) was proven to be cytotoxic in the dilutions 100 to 8.8% V/V. The assay fulfilled all acceptance criteria.

The quantification of the viability of the cells after 24 ± 2 h incubation with the test item showed a minimal viability of 84.1% in the dilution of 7 mM and a grading of "0" for all concentrations tested. In conequence, no cytotoxic effect was demonstrated for SP02.