Poly(oxy-1,2-ethanediyl), α,α'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

EC Number:

615-231-8

Cas Number:

70983-58-3

Molecular formula:

UVCB

IUPAC Name:

Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

Test material form:

liquid: viscous

Details on test material:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Specific details on test material used for the study:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

In vitro test system

Details on the study design:

The KeratinoSens assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.

Preparation of the Test Item

All test item solutions were freshly prepared immediately prior to use.
Since the test item had no defined molecular weight, the test was performed using a pro forma molecular weight of 200 g/mol. A stock solution with a concentration of 40 mg/mL was prepared by pre-weighing the test material into a glass vial and dissolving the test item in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%, AppliChem, Lot. No.: 0000978834).
Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
 
Controls

A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Cell line

The test was carried out using the transgenic cell line KeratinoSens (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 3 experiment 1 and 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +- 1°C and 5% CO2. For test material exposure, cells were cultured in medium.

Composition of Media

Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
- 1% geneticin (final concentration: 500 µg/mL)

Assay Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1879620, 1880290) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
 
Test Item Exposure Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX) with 1.0 g/L D-glucose and Na-Pyruvate.
The medium was supplemented with the following components:
- 1% fetal bovine calf serum

Luciferase Assay System

The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual.
Luciferase Assay System 10-Pack.

The kit consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer

If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent

Prior to use lysis buffer was diluted 1:5 with dist. water.
10.7. Further Reagents

MTT solution
- MTT stock solution: 5 mg/mL MTT in DPBS

SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS) in dist. water

DPBS:
DPBS solution (without Ca2+/Mg2+)

Dose Groups
1.Negative Control: DMSO: 1% (v/v) in test item exposure medium
2.Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3.Test Item: 12 concentrations of the test item

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

Experimental Procedure

A cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1866243, 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Calculation of EC1.5, IC50 and IC30, Cell Viability and Maximal Induction of the Luciferase Activity was performed according to OECD 442 d guideline.

Prediction Model

The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 200 µg/mL
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <200 µg/mL is considered as inconclusive.

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Results and discussion

Positive control results:

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: EC1.5

Remarks:

[µM]

Value:

6.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: Imax

Value:

1.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: IC30

Remarks:

[µM]

Value:

7.77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: IC50

Remarks:

[µM]

Value:

10.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Run / experiment:

other: positive control - experiment 1

Parameter:

other: EC1.5

Remarks:

PC

Value:

24.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Run / experiment:

other: positive control - experiment 2

Parameter:

other: EC1.5

Remarks:

PC

Value:

8.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results

The in vitro KeratinoSens assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test item was dissolved in DMSO. Since the test item had no defined molecular weight, the test was be performed using a pro forma molecular weight of 200 g/mol. Based on this, a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium.

The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.80 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 75.9%. No further significant luciferase induction was observed. The calculated EC1.5 was < 200 µg/mL (6.35 µM corresponding to 1.27 µg/mL). In the second experiment, a max luciferase activity (Imax) induction of 1.87 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 58.0%. No further significant luciferase induction was observed. The calculated EC1.5 was < 200 µg/mL (6.21 µM corresponding to 1.24 µg/mL). No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.29 µM in experiment 1; 8.67 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (9.6% in experiment 1; 10.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled. In the second experiment the threshold was slightly increased with a fold induction of 8.59. As in both experiments the values of the test item were very comparable and all other acceptance criteria were met, this slight increase was accepted.

Table 1: Results of the cytotoxicity measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	100	100	100	-
Positive control	4.00 8.00 16.00 32.00 64.00	100.7 104.3 106.8 118.1 122.0	99.7 101.3 109.4 119.4 132.6	100.2 102.8 108.1 118.7 127.3	0.7 2.1 1.8 0.9 7.5
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	98.9 114.0 115.2 75.9 15.9 0.2 0.4 0.8 1.1 1.3 1.6 1.5	106.2 108.4 113.3 58.0 1.3 0.3 0.5 0.7 0.8 1.2 1.3 1.1	102.6 111.2 114.3 66.9 8.6 0.2 0.4 0.7 1.0 1.2 1.4 1.3	5.2 3.9 1.3 12.6 10.3 0.0 0.1 0.0 0.2 0.1 0.2 0.3

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.08 1.18 1.34 1.58 2.39	1.13 1.14 1.30 1.61 2.60	1.21 1.25 1.40 1.73 2.66	1.14 1.19 1.35 1.64 2.55	0.07 0.06 0.05 0.08 0.14	* *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.99 0.78 0.98 1.53 1.45 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.87 0.81 1.08 1.92 0.97 0.00 0.00 0.00 0.00 0.00 0.02 0.00	0.88 0.75 0.92 1.96 1.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.91 0.78 0.99 1.80 1.22 0.00 0.00 0.00 0.00 0.00 0.01 0.00	0.07 0.03 0.08 0.24 0.24 0.00 0.00 0.00 0.00 0.00 0.01 0.00	*

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.18 1.37 1.76 3.11 6.71	1.42 1.41 2.08 3.79 9.59	1.01 1.59 2.05 3.43 9.47	1.20 1.46 1.96 3.44 8.59	0.20 0.12 0.18 0.34 1.63	* * *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.93 0.87 0.84 1.37 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.84 0.86 0.91 2.01 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.89 1.01 1.14 2.24 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.89 0.91 0.96 1.87 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.04 0.09 0.16 0.45 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.14 1.19 1.35 1.64 2.55	1.20 1.46 1.96 3.44 8.59	1.17 1.32 1.66 2.54 5.57	0.04 0.19 0.44 1.27 4.27
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.91 0.78 0.99 1.80 1.22 0.00 0.00 0.00 0.00 0.00 0.01 0.00	0.89 0.91 0.96 1.87 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.90 0.85 0.98 1.84 0.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.02 0.09 0.02 0.05 0.77 0.00 0.00 0.00 0.00 0.00 0.01 0.00	*

* = significant induction according to Student’s t-test, p<0.05

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In this KeratinoSenss tudy under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.