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Administrative data

Description of key information

The sensitizating potential was tested in three vitro studies (Myeloid U937 Skin Sensitization Test (MUSST), in ARE Reporter Assay (LuSens) and Direct Peptide Reactivity Assay (DPRA). The test item did reveal sensitising properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Principles of method if other than guideline:
In vitro Sensitization: Direct Peptice Reactivity Assay (DPRA)
GLP compliance:
yes
Remarks:
non-GLP, but study conducted under similar Quality assurance system.
Type of study:
other: Direct Peptice Reactivity Assay (DPRA)
Details on study design:
The test substance was solved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity.

The study is performed according to the methods described in the following publications:
Bauch C, Kolle SN, Ramirez-Hernandez T, Eltze T, Fabian E, Teubner W, Mehling A, van Ravenzwaay B, Landsiedel R. Putting the parts together: Combining in vitro methods to test for skin sensitizing. Regulatory Toxicology and Pharmacology 63(3), 489-504, 2012. 2 Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. Quantification of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Cysteine-Peptide
Value:
30.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: moderate reactivity
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Lysine-Peptide
Value:
2.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: minimal reactivity
Parameter:
other: mean peptide depletion [%]
Run / experiment:
mean of both depletions
Value:
16.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: low reactivity

The test substance was solved in acetonitrile. However, when mixed with the peptide stock solutions the samples became opaque. Visual observation after the 24-hour incubation time revealed precipitates in all samples. Based on the observed results and applying the prediction model proposed in Geberick et. al (2007) it was concluded that Laurylacrylate 1214 shows low chemical reactivity in the DPRA under the conditons chosen.

Interpretation of results:
other: low chemical reactivity
Conclusions:
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro sensitization: Dendritic Cell Line Activation Assay; Myeloid U 937 Skin Sensitization Test (MUSST)
GLP compliance:
yes
Remarks:
non-GLP, but study conducted under similar Quality assurance system.
Type of study:
other: In vitro sensitization: Dendritic Cell Line Activation Assay; Myeloid U 937 Skin Sensitization Test (MUSST)
Details on study design:
- Cell line U937
- Vehicle: culture medium
- The myeloid U937 skin sensitization test is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate fer dendritic cells. As readout, the change in the expression of the cell membrane marker GD 86 measured by Iow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when GD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments.
- Positive control substance: Ethylene diamine (EDA – 70 μg/mL)
- The cytotoxicity of the test substance was evaluated by flow cytometry using propidium iodide staining after 48 hours exposure. For the purpose the CV75 value was derived from the concentration response curve. The CV75 is the estimated concentration that affords 75% cell viability and was determined to be 1297.8 µg/mL for Laurylacrylate 1214 under the chosen exposure conditions on U937 cells. In the main test, test substance was assessed at six final concentrations 2595.6 µg/mL, 1297.8 µg/mL, 648.9 µg/mL, 350.0 µg/mL and 324.0 µg/mL to be 162.2 µg/mL for Laurylacrylate 1214 under the chosen exposure conditions on U937 cells.
After 48 hours of exposure U937 cells were stained with FITC labeled anti-human-CD 86 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently non-cytotoxic (cell viability >/= 70%) concentration in two experiments.
The strong sensitizer ethylene diamine (EDA, 70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control.
Parameter:
other: CD86 fold induction
Run / experiment:
162.2 µg/mL, 1st Experiment
Value:
1.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
324.5 µg/mL, 1st Experiment
Value:
1.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
350.0 µg/mL, 1st Experiment
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not measured/tested
Parameter:
other: CD86 fold induction
Run / experiment:
648.9 µg/mL, 1st Experiment
Value:
1.32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
1297.8 µg/mL, 1st Experiment
Value:
1.49
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
2595.6 µg/mL, 1st Experiment
Value:
1.52
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
162.2 µg/mL, 2nd Experiment
Value:
0.93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
324.5 µg/mL, 2nd Experiment
Value:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
350.0 µg/mL, 2nd Experiment
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not measured/tested
Parameter:
other: CD86 fold induction
Run / experiment:
648.9 µg/mL, 2nd Experiment
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
1297.8 µg/mL, 2nd Experiment
Value:
0.54
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
2595.6 µg/mL, 2nd Experiment
Value:
0.91
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
162.2 µg/mL, 3rd Experiment
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not measured/tested
Parameter:
other: CD86 fold induction
Run / experiment:
324.5 µg/mL, 3rd Experiment
Value:
0.94
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
350.0 µg/mL, 3rd Experiment
Value:
0.98
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
648.9 µg/mL, 3rd Experiment
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
1297.8 µg/mL, 3rd Experiment
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not measured/tested
Parameter:
other: CD86 fold induction
Run / experiment:
2595.6 µg/mL, 3rd Experiment
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not measured/tested

In summary, after 48 hours of exposure to test substance Laurylacrylate 1214 CD 86 expression was not induced in U937 cells at concentration between 162.2 to 2595.6 µg/mL affording at least 70% viability. From this it has to be concluded that test substance Laurylacrylate 1214 does not induce dendritic cell activation.

Interpretation of results:
other: no induction of dendritic cell activation
Conclusions:
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
In summary, after 48 hours of exposure to the test substance CD86 expression was not induced in U937 cells at concentration between 162.2 to 2595.6 µg/mL affording at least 70 % viability in more than one experiment. From this it has to be concluded that the test substance does not induce dendritic cell activation.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro Test LuSens (Bauch et al. 2012)
GLP compliance:
yes
Remarks:
non-GLP, but study conducted under similar Quality assurance system.
Type of study:
other: in vitro LuSens
Details on study design:
The cell line LuSens was treated with 6 test substance concentrations for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition (Steady Glo®, Promega). ln parallel a MTT assay was performed to assess cytotoxicity of the test substance. A test substance was considered to have an ARE induction potential if the fold induction of luciferase activity was > 1.5 and viability determined in the MTT assay was > 70% at any test concentration.

MTT assay:
The assay is based on the tetrazolium salt MTT. ln active mitochondria the tetrazolium ring of yellow MTT is cleaved by various dehydrogenase enzymes and a purple formazan is formed. Gell culture medium was aspirated from all wells and 180 ~L medium 3 was added. Briefly, a 5 mg/mL thiazolyl blue tetrazolium bromide (MTT) stock solution was prepared in PBS (without Ca2+/Mg2+). 20 µL of MTT solution will be added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis medium was aspirated and cells lysed by adding 100 ~L of Iysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SOS; and 0.4 ml glacial acetic acid). Absorbance was
measured at 570 nm with reference wavelength at 590 nm 690 nm using the Sunrise TM Absorbance Reader.

Luciferase assay:
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+ /Mg2+), 100 µL Steady-Glo- Mix and 100 µL PBS (without Ca2+ /Mg2+) per weil were added and cells shaked with a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in a luminometer.
Parameter:
other: luciferase activity fold induction
Run / experiment:
6.82 µg/mL, 1st Experiment
Value:
4.29
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
8.18 µg/mL, 1st Experiment
Value:
4.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
9.81 µg/mL, 1st Experiment
Value:
4.71
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
11.78 µg/mL, 1st Experiment
Value:
4.47
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
14.13 µg/mL, 1st Experiment
Value:
4.01
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
16.96 µg/mL, 1st Experiment
Value:
2.81
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
6.82 µg/mL, 2nd Experiment
Value:
8.09
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
8.18 µg/mL, 2nd Experiment
Value:
9.58
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
9.81 µg/mL, 2nd Experiment
Value:
8.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
11.78 µg/mL, 2nd Experiment
Value:
6.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
14.13 µg/mL, 2nd Experiment
Value:
4.59
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
16.96 µg/mL, 2nd Experiment
Value:
4.57
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

ln summary, after 48 hours of exposure to test substance Laurylacrylate 1214 luciferase activity in LuSens cells was induced at concentration between 6.82 and 9.81 µg/mL affording at least 70% viability. From this it has to be concluded that test substance Laurylacrylate 1214 has an keratinocyte activating potential.

Interpretation of results:
other: keratinocyte activating potential
Conclusions:
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced at concentration between 6.82 and 9.81 µg/mL affording at least 70 % viability. From this it has to be concluded that the test substance has an keratinocyte activating potential.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key steps of the sensitization process: protein reactivity (DPRA), activation of keratinocytes (LuSens or KeratinoSens) and activation of dendritic cells (MUSST or h-CLAT) has been used. At the time this test battery evaluation report was written, the in vitro methods were used to assess the sensitization potential.
GLP compliance:
yes
Remarks:
non-GLP, but study conducted under similar Quality assurance system.
Type of study:
other: in vitro test battery
Details on study design:
Details of the study procedures are described in the individual projects in the respective reports (and study plans for studies conducted according to GLP).
- Direct Peptide Reactivity Assay (DPRA): 64V0545/22A322
- Keratinocyte Activation Assay (LuSens): 66V0545/11A324
- Dendritic Cell Line Activation Assay, Myeloid U937 Skin Sensitization Test (MUSST): 65V0545/11A323

Test battery evaluation
The test battery evaluation uses the results of three individual assays testing reflecting three key events along the adverse outcome pathway (AOP) leading to skin sensitization: peptide reactivity (DPRA), keratinocyte activation (LuSens or KeratinoSens) and dendritic cell activation (MUSST or h-CLAT).
Each individual assay was validated using 54 substances. The results were then combined in a test battery evaluation to qualitatively assess the skin sensitizing potential (hazard) of the test substance (Bauch et al., 2012).
In the test battery evaluation a weight of evidence (WoE) approach is used: Any two of the three tests determine the overall results, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al., 2012).
Parameter:
other: mean peptide depletion [%]
Run / experiment:
DPRA
Value:
16.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: luciferase activity fold induction
Run / experiment:
LuSens
Value:
> 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 fold induction
Run / experiment:
MUSST
Value:
< 1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results and applying the evaluation criteria, the test substance is predicted to be a skin sensitizer.
Executive summary:

A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test substance.

- proteine reactivity (DPRA),

- activation of keratinocytes (LuSens), and

- activation of dendritic cells (MUSST).

In this report, the results of the individual studies are summarized and evaluated to predict the presence or absence of skin sensitizing potential of the test substance.

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD Test Guideline No. 429 (Local Lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95 % compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86 %).

A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing this report.

Based on the results and applying the evaluation criteria, the test substance is predicted to be a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitizing potential of the read across substance 2-Propenoic acid, C12-14-alkyl esters was investigated in three in vitro testes:

MUSST:

The read across substance 2-Propenoic acid, C12-14-alkyl esters was tested in the myeloid U937 skin sensitization test. The test is performed using the human pro-monocytic cell line U937 as surrogate fer dendritic cells. As readout, the change in the expression cf the cell membrane marker GD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when GD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments. After 48 hours of exposure to test substance Laurylacrylate 1214 CD 86 expression was not induced in U937 cells at concentration between 162.2 to 2595.6 µg/mL affording at least 70% viability in more than one experiment From this it has to be concluded that test substance Laurylacrylate 1214 does not induce dendritic cell activation.

DPRA:

The read across substance 2-Propenoic acid, C12-14-alkyl esters was tested in the Direct Peptide Reactivity Assay (DPRA). The test substance was solved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity. The test substance was solved in acetonitrile. However, when mixed with the peptide stock solutions the samples became opaque. Visual observation after the 24-hour incubation time revealed precipitates in all samples. Based on the observed results and applying the prediction model proposed in Geberick et al. (2007) it was concluded that Laurylacrylate 1214 shows low chemical reactivity in the DPRA under the conditons chosen.

Lu Sens:

The read across substance 2-Propenoic acid, C12-14-alkyl esters was tested in the LuSens assay.

A luciferase reporter cell line (LuSens cells) based on the activation of the antioxidant response element that can be used to assess the keratinocyte activating potential of a substance. The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012). lt employs the reporter gene for luciferase under the control of an ARE and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with test substances. after 48 hours of exposure to test substance Laurylacrylate 1214 luciferase activity in LuSens cells was induced at concentration between 6.82 and 9.81 µg/mL affording at least 70% viability. From this it has to be concluded that test substance Laurylacrylate 1214 has an keratinocyte activating potential.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008 (GHS Category 1, H317), as amended for the tenth time in Regulation (EC) No. 2017/776. A subcategorization for potency is not possible from the existing experimental data.