Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-06-30 to 2015-08-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study follows published method considered scientifically valid by ECVAM

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this in vitro study the embryotoxic/teratogenic potenitial of the test item was investigated using mouse embryonic stem cells in the µEST test. Furthermore, the cytotoxicity of the compound was measured in the same cell line (XTT test).

For the μEST FL, embryonic stem cells were differentiated towards cardiac tissue in the presence or absence of the test compound. The compound was tested at 5 different concentrations in 4 individual experiments. Cell type specific expression of the fluorescent reporter protein allows to quantify the amount of cardiac cells. Differences in fluorescence intensity between treated cells and untreated controls were used to determine an embryotoxic and/or teratogenic effect using a prediction model.
For the cytotoxicity test, embryonic stem cells were cultured in 3 individual experiments on plates and treated with 6 concentrations of the test compound. The conversion of XTT in viable cells into a water-soluble formazan product was used as read-out.
For both tests, a vehicle control and a positive control were run concurrently.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
developmental toxicity / teratogenicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Storage condition of test material: stored at room temperature

Test animals

Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Administration / exposure

Vehicle:
DMSO
Details on exposure:
PREPARATION OF TEST COMPOUND:
- on each experimental day, an aliquot of the compound was weighed and diluted in the solvent to a stock concentration of 0.01 M.
- consecutive 10-fold dilutions were performed in the appropriate cell culture medium to reach the desired concentrations.

The final concentration of solvent was <0.1 % for all experiments.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
µEST FL test (embryotoxicity): 14 days
XTT test (cytotoxicity): 10 days
Frequency of treatment:
µEST FL test (embryotoxicity): treatment on days 1 and 7
XTT test (cytotoxicity): treatment on days 2, 4 and 7
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10^-5 M, 10^-6 M, 10^-7 M, 10^-8 M, and 10^-9 M
Basis:
nominal conc.
µEST FL test
Remarks:
Doses / Concentrations:
10^-5 M, 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, and 10^-10 M
Basis:
nominal conc.
XTT test
No. of animals per sex per dose:
three replicates of each treatment
Control animals:
other: control (DMSO) cell cultures were used
Details on study design:
TEST SYSTEM:
- cell system: mouse embryonic stem (ES) cell clone aMHC 23 (specific)
- aMHC clone 23 is a mouse ES cell clone (origin: D3) stably transfected with a GFP reporter gene construct under control of the cardiac-specific aMHC promoter (Kolossov et al., 2005)*.
- aMHC clone 23 ES cells are cultured on a layer of irradiated mouse embryonic fibroblasts (MEFs) in tissue culture dishes in DMEM 15% FBS.
- Leukemia inhibitory factor (LIF) is added at to ensure the undifferentiated phenotype.
- cells are trypsinized and replated every 48 hours (cell density: about 2.5 x 10^5 cells/dish).
- at least 3 passages are performed before the cells are used in the μEST FL test.


µEST FL TEST (embryotoxicity):
Day 0:
- ES cells (2 x 10^6/ mL) are aggregated to embryoid bodies (EBs) in differentiation medium (IMDM + 20 % FCS; 37 °C, 5 % CO2, 95% humidity) in dishes on a horizontal shaker for 6 hours to start differentiation.
- suspension is then diluted 1:20 in differentiation medium into culture flasks and incubate on the shaker overnight.

Day 1:
- approx. 35 EBs are diluted into each well of 6-well plates containing differentiation medium
- test compound (1:100 of the prediluted compound) are added.
- test compound is tested in 5 dilutions in triplicates in 4 independent experiments.
- vehicle control was also performed.
- EBs are cultured at 37 °C, 5% CO2, 95% humidity.

Day 7: differentiation medium with fresh test compound is added.

Day 14:
- EBs from each well are counted, transferred into tubes, washed with PBS and lysed with lysis buffer.
- lysates are transferred to plates
- fluorescence is measured using a fluorescence spectrophotometer (green fluorescent protein (GFP) at 476/510 nm, background at 476/535 nm).

XTT TEST (cytotoxicity):
Day 1:
- ES cells are seeded in gelatine-coated 96-well plates (2000 cells/well) in culture medium and cultured overnight (37 °C, 7 % CO2 and 95 % humidity).

Day 2:
- double-concentrated compound solution is added per well (six 10-fold dilutions in hexaplicates, 1 column/compound dilution).
- two columns are used for vehicle control.
- cells are incubated at 37 °C, 7% CO2 and 95% humidity.

Day 4 and Day 7:
- complete change of medium, adding of fresh compound.

Day 10:
- incubation with XTT for 50 minutes at 37 °C, adding of stop solution, measurement of absorbance at 475 nm.


DATA ANALYSIS - µEST FL Test:
1. The values of the fluorescence intensity with extinction at 476 nm and emission at 510 nm are corrected for autofluorescence of cell lysates at emission 535 nm.

2. Fluorescence values have to be normalized with respect to EB number. Therefore, measurement values are divided by the number of EBs in the corresponding well of the test plates to standardize the values to ”fluorescence intensity per EB”.

3. For each set of data, the mean value of all associated control values obtained in the same experiment (”run”) is calculated (“run control mean”).

4. All values obtained in 2. are divided by the run control mean to create effect ratios. Values measured in different runs pertaining to one compound and one concentration are thus made comparable. Subsequently, the mean value of all effect ratios is computed and converted to percentage values by multiplication with 100.

5. The calculation of errors (standard error of the mean) is performed on all effect ratios from all runs of a compound testing. The percentage error is calculated by multiplication with 100.

6. Initially, dose effects are given for measured concentrations only. These values have to be linearly interpolated between adjacent points to form a continuous dose-response curve. To calculate EC50 and EC25 (ECx ), the first measured concentration – starting from the lowest – to exceed an effect of x % (i.e. to fall below the (100-x) % level) is determined (please refer to the field "Statistics" below). In case of the curve to exceed an effect of x % at the lowest measured concentration, this concentration is assumed to be the ECx value. If an effect of x % is never reached within the measured concentration range, the ECx value cannot be determined.

7. To calculate the significance of the difference from the control value, a one-sample t-test is performed. The values being used are the mean of differences, the standard deviation and the sample size.

DATA ANALYSIS - XTT Test:
1. The absorption values measured at the reference wavelength (630 nm) are subtracted from the values measured at the specific wavelength (475 nm).

2. For each plate (= one compound), the mean value of the blank measurement values is calculated (”blank mean”) and subtracted from all other values. Subsequent calculations are performed with blank-corrected values.

3. For each plate (= one compound), the mean value of the vehicle controls is calculated (”control mean”).

4. All measurement values are divided by the control mean to create effect ratios. Values measured in different runs pertaining to one compound and one concentration are thus made comparable. Subsequently, the mean value of all these effect ratios is computed and converted to percentage values by multiplication with 100.

5. Calculation of errors and of inhibitory concentrations (ICx ) are performed as described in steps 5.-7. above.


DATA INTERPRETATION:
For interpretation of the data, four different cases can be distinguished as follows:
- case 1: neither with μEST FL nor with XTT an effective concentration is reached: The compound is without toxicological findings.
- case 2: with μEST FL, an EC50 is reached, or a drop out of a 100 ± 25 % corridor (EC25) at two consecutive concentrations: The compound is “embryotoxic”.
- case 3: with XTT, an IC50 is reached, or a drop out of a 100 ± 25% corridor (IC25) at two consecutive concentrations: The compound is definitely “generally toxic”.
- case 4: in both methods an EC/IC50 or a drop out of a 100 ± 25 % corridor (EC/IC25) at two consecutive concentrations is reached: In this case the concentration range in which the effect occurs determines the classification. The cut-off concentration was empirically determined during validation of the method.
case 4a: if an ECx is reached at a concentration below 5 X 10^-6 M, the compound is classified as ”embryotoxic”
case 4b: if both effects are reached at concentrations above 5 X 10-6 M, the compound is classified as ”generally toxic”

CALCULATION OF EC50 AND IC50
1. Let f be the function of effect in percent to a given dose. Let c1, c2 be two consecutive concentrations with c1 < c2 and f (c1) > 50% and f (c2) ≤ 50 %.

2. Then the EC50 or IC50 value within the interval [c1, c2] is determined by the condition f (EC/IC50) = 50 %. The intersection point is calculated by linear interpolation on a logarithmic concentration scale.

3. In μEST FL, an embryotoxic/teratogenic effect can also be detected by an increase of GFP values (dropping out of a 100 ± 50 % corridor). Therefore, the condition for the EC50 value within the interval [c1, c2] has to be modified to |f (EC50) − 100 %| = 50 %.

4. If all values lie within the 100 ± 50 % corridor, it is not possible to determine an EC50 or IC50. (In particular, no extrapolation is performed.)

5. If the value at the lowest measured concentration lies outside of the 100 ± 50 % corridor, this concentration is assumed to be the IC50 or EC50, respectively.

*Reference:
- Kolossov E, Lu Z, Drobinskaya I, et al. 2005. Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes. FASEB. J. 19(6): 577-9.

Examinations

Examinations:
not applicable
Positive control:
The known embryotoxic compound retinoic acid was used as positive control in the µEST FL test and the XTT test (concentrations: 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, and 10^-10 M as well as 10^-11 M (XTT test only)) and was prepared as follows:
- positive control was dissolved in DMSO at 0.001 M and stored in 50 μL aliquots at -20 °C.
- on each experimental day, one aliquot was thawed and further diluted in the appropriate cell culture medium to the desired concentrations.

Results and discussion

Details on results:
RESULTS OF THE TEST ITEM:
Case 1: not embryotoxic

µEST test:
- the compound did show a slight effect only in one run. This effect was reversely dose-dependent, and considered to be due to technical issues.
- the average of the test runs, which is used for the final assessment, does not show any effect on differentiation.

XTT test:
- one run showed a dose-dependent cytotoxic effect of the compound.
- two further runs did not show any cytotoxic effect at all.
- in the run showing a dose-dependent cytotoxic effect of the compound, even the second column of vehicle control wells, which are in direct contact with the highest compound concentration, did show an decrease in cell viability which was similar to the effect of the highest compound concentration, suggesting diffusion of the compound through the plastic of the wells.
- a fourth run could not be measured since the activated XTT solution instantly turned brownish after it was added to the plates.

Overall, the compound does not exert an effect on differentiation in the μEST assay and is therefore considered to be not embryotoxic under the conditions of the assay. The compound does show a cytotoxic effect in 1 out of 3 XTT tests; therefore, we can’t exclude a general cytotoxic effect. However, the classification regarding the embryotoxicity would not change even if the compound is considered to be cytotoxic, since no effect on differentiation could be seen.


RESULTS OF THE POSITIVE CONTROL:
Case 4a: embryotoxic

µEST test:
- the compound exerts a strong effect on differentiation.
- no differentiation was detected up to 1e-10 M

XTT test:
- the compound exerts a strong effect on the cell’s viability.
- the cytotoxic effect is still pronounced both at 1e-9M and 1e-10M.

Overall, the compound is considered to be embryotoxic according to the prediction model.

In one run of the test on differentiation, the last 2 concentrations are missing, since no EBs survived in these wells. This may have a similar as stated for the same run with the test compound.

The one run of the XTT test could not be measured, since the activated XTT solution instantly turned brownish after it was added to the plates. The cytotoxicity test therefore consists of three test runs. In the second XTT test 2 concentrations are missing due to a pipetting error.

Please also refer to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

Table 1: Effect of the test item on differentiation of specific cell type (μEST FL 14d treat.)

Concentration [M]

N

Mean [%]

SEM [%]

p

10-9

5

84

8.5

0.066

10-8

10

82.5

6.4

0.011

10-7

10

88.6

8.9

0.116

10-6

12

90

6.6

0.077

10-5

8

106.1

12.2

0.317

EC50:∞ M

EC25:∞ M

Table 2: Effect of the test item on proliferation and survival of specific cell type (XTT 10d treat.)

Concentration [M]

N

Mean [%]

SEM [%]

p

10-10

17

97.9

4.6

0.327

10-9

17

90

3.8

0.009

10-8

17

99.9

3.8

0.487

10-7

18

93.7

8.4

0.231

10-6

18

77.5

11.3

0.032

10-5

18

70.6

10.4

0.006

IC50:∞ M

IC25: 2.31 x10-6

Table 3: Effect of the positive control on differentiation of specific cell type (μEST FL 14d treat.)

Concentration [M]

N

Mean [%]

SEM [%]

p

10-10

4

7.9

1.2

0

10-9

5

4

0.6

0

10-8

8

7.5

1.9

0

10-7

8

5.4

1.5

0

10-6

8

5.1

1.4

0

EC50:10-10M

EC25:10-10 M

Table 4: Effect of the positive control on proliferation and survival of specific cell type (XTT 10d treat.)

Concentration [M]

N

Mean [%]

SEM [%]

p

10-11

12

59.2

4.4

0

10-10

6

71.7

3

0

10-9

6

50.9

4.8

0

10-8

12

44.8

1.9

0

10-7

11

41.2

2.7

0

10-6

12

35.2

4.9

0

IC50:1.39 x  10-9M

IC25:10-11M

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this in vitro study, the test item is considered to be not embryotoxic according to the prediction model. Furthermore, a dose-dependent cytotoxic effect was observed, but this effect does not affect the prediction of the test compound to be not embryotoxic.
Executive summary:

In this in vitro study the embryotoxic/teratogenic potenitial of the test item was investigated using the µEST FL test. For the μEST FL, mouse embryonic stem cell clone aMHC 23 (specific) were differentiated towards cardiac tissue in the presence or absence of the test compound. The compound was tested at 5 different concentrations (10-5M,10-6M,10-7M,10-8M, and10-9M) in 4 individual experiments. Cell type specific expression of the fluorescent reporter protein allows to quantify the amount of cardiac cells. Differences in fluorescence intensity between treated cells and untreated controls were used to determine an embryotoxic and/or teratogenic effect using a prediction model.

Furthermore, the cytotoxicity of the compound was measured in the XTT test using the same cell line. For the cytotoxicity test, embryonic stem cells were cultured in 3 individual experiments on plates and treated with 6 concentrations of the test compound (10-5M,10-6M,10-7M,10-8M,10-9M, and10-10M) . The conversion of XTT in viable cells into a water-soluble formazan product was used as read-out.

For both tests, a vehicle control and a positive control were run concurrently.

The test item is considered to be not embryotoxic according to the prediction model. Furthermore, a dose-dependent cytotoxic effect was observed, but this effect does not affect the prediction of the test compound to be not embryotoxic.

Categories Display