(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Endpoint summary

Description of key information

In an integrated approach adressing key events of the skin sensitisation AOP, results from an in chemico skin sensitisation study according to OECD guideline 442 C (DPRA) and the in vitro ARE-Nrf2 Luciferase Test Method (KeratinoSens, OECD guideline 442 D) were combined and both predicted a non-skin sensitising potential of the test item (reference 7.4.1-1 and 7.4.1-2).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Justification for non-LLNA method:

This ARE-Nrf2 luciferase test method can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), DPRA (direct peptide reactivity assay)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Details on the study design:

PREPARATION OF TEST ITEMS
- Test item concentrations: 0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM

PREPARATION OF POSITIVE/NEGATIVE CONTROLS
- Positive control: 4 µM - 64 µM cinnamic aldehyde
- Solvent (test item + postive control): 1% (v/v) DMSO
- Negative Control: 1% (v/v) DMSO

EXPERIMENTAL PROCEDURE
- Incubation: Cells were grown for 24h ± 1h in assay medium at 37°C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 200 µL test item in exposure medium and incubated for 48h ± 1h.

- Measurement of luciferase activity: Cells were washed once with DPBS and 20 µL of passive lysis buffer was added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates were placed in the plate reader for luminescence measurement. 50 µL/well of the luciferase substrate was injected. The plate reader waited for 1 sec before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

- Cell viability: 2.7 mL of a MTT solution (5 mg/mL in DPBS) was added to 20 mL exposure medium. The medium was replaced with 200 pL of this fresh medium containing MTT. The plate was covered with a sealing tape and incubated for 4h 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% C02 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at X = 600 nm.

REPLICATES
Two independent repetitions. Each independent run consisted of three replicates for each concentration step of the test item and the positive control.

Key result

Run / experiment:

other: Run 2

Parameter:

other: Dose-response for luciferase induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 1

Parameter:

other: Dose-response for luciferase induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 2

Parameter:

other: EC1.5 value < 1000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 1

Parameter:

other: EC1.5 value < 1000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 2

Parameter:

other: Cell viability > 70% at lowest concentration with an induction of luciferase activity > 1.5-fold

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 1

Parameter:

other: Cell viability > 70% at lowest concentration with an induction of luciferase activity > 1.5-fold

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Run 2

Parameter:

other: Imax > 1.5-fold increased

Value:

1.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Run 1

Parameter:

other: Imax > 1.5-fold increased

Value:

0.96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table 1: Cytotoxicity

  

	Concentration [uM]	Cell Viability [%]
		Run 1	Run 2
Solvent Control	-	100	100
	4	105.90	105.80
	8	114.68	112.51
Positive Control	16	132.81	126.61
	32	134.96	132.24
	64	124.17	139.76
	0.98	95.68	107.94
	1.95	99.86	102.04
	3.9	89.21	96.40
	7.8	88.92	90.22
	15.6	72.37	95.46
Test Item	31.25	89.93	97.47
	62.5	86.76	96.13
	125	89.35	99.08
	250	76.55	97.74
	500	72.81	95.32
	1000	79.57	93.58
	2000	77.12	91.16

 

 

Table 2: Luciferase activity run 1

 

  

	Concentration [uM]	Cell Viability [%]
		Run 1	Run 2
Solvent Control	-	100	100
	4	105.90	105.80
	8	114.68	112.51
Positive Control	16	132.81	126.61
	32	134.96	132.24
	64	124.17	139.76
	0.98	95.68	107.94
	1.95	99.86	102.04
	3.9	89.21	96.40
	7.8	88.92	90.22
	15.6	72.37	95.46
Test Item	31.25	89.93	97.47
	62.5	86.76	96.13
	125	89.35	99.08
	250	76.55	97.74
	500	72.81	95.32
	1000	79.57	93.58
	2000	77.12	91.16

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

 

 

Table 3: Luciferase activity run 2

                  

	Concentration [uM]	Fold Induction				Significance
		Rep. 1	Rep. 2	Rep. 3	Mean
Solvent Control	-	1.00	1.00	1.00	1.00
Positive Control	4	1.13	1.17	1.18	1.16
	8	1.36	1.27	1.26	1.30
	16	1.63	1.48	1.48	1.53	*
	32	2.81	1.98	2.43	2.41	*
	64	5.72	5.28	5.58	5.53	*
Test Item	0.98	0.88	0.95	0.97	0.93
	1.95	1.04	0.90	0.99	0.98
	3.9	1.00	0.95	0.96	0.97
	7.8	1.01	0.92	1.01	0.98
	15.6	0.97	0.80	0.97	0.91
	31.25	1.00	1.08	0.95	1.01
	62.5	1.09	0.82	0.97	0.96
	125	0.96	0.80	1.13	0.96
	250	1.04	0.74	1.01	0.93
	500	1.12	0.85	1.02	1.00
	1000	1.15	0.93	1.15	1.08
	2000	1.15	0.96	0.99	1.03

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

 

 

Table 4: Luciferase activity - overall induction

 

	Concentration	Fold Induction
	[uM]	Run 1	Run 2
Solvent Control	-	1.00	1.00
	4	1.19	1.16
	8	1.54	1.30
Positive Control	16	2.11	1.53
	32	4.47	2.41
	64	28.92	5.53
	0.98	0.87	0.93
	1.95	0.84	0.98
	3.9	0.91	0.97
	7.8	0.90	0.98
	15.6	0.96	0.91
Test Item	31.25	0.76	1.01
	62.5	0.76	0.96
	125	0.70	0.96
	250	0.79	0.93
	500	0.75	1.00
	1000	0.86	1.08
	2000	0.81	1.03

 

 

Table 5: Additional parameters

	Run 1	Run2	Mean
EC1.5 [uM]	n.d.	n.d.	n.d.
imax	0.96	1.08	1.02
IC30 [uM]	n.d.	n.d.	n.d.
IC50 [uM]	n.d.	n.d.	n.d.

n.d. = cannot be determined

 

 

Table 6: Acceptability of the test

Run 1;

 

Acceptance Criterion	Result
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.	8 uM: 1.54 16 uM: 2.11 32 uM: 4.47 64 uM: 28.92	Pass
The average induction in the three technical replicates for the positive control at a concentration of 64 uM is between 2 and 8.	28.92	Pass*
The ECi 5 value of the positive control is within two standard deviations of the historical mean (1.33 uM - 31.19 uM based on the historical data of the testing laboratory).	7.54 uM	Pass
The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control is < 20% in each repetition which is consisting of 6 wells.	18.7%	Pass
* If this criterion is not fulfilled, the dose-response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control according to OECD 442D. A clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control was observed (see part 11.2) and, thus, this run will be accepted. Run 2:
Acceptance Criterion	Result
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.	16 pM: 1.53 32 uM: 2.41 64 pM: 5.53	Pass
The average induction in the three technical replicates for the positive control at a concentration of 64 uM is between 2 and 8.	5.53	Pass
The ECi 5 value of the positive control is within two standard deviations of the historical mean (1.33 uM - 31.19 uM based on the historical data of the testing laboratory).	14.96 uM	Pass
The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control is < 20% in each repetition which is consisting of 6 wells.	13.9%	Pass

 

The study met all acceptance criteria

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, the test item did not induce 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.

Executive summary:

The test item was completely dissolved in treatment culture medium up to a concentration of 2000 µM. In the first run, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. In the second run, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. 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 data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-09 to 2020-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted 2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Details on the study design:

PREPARATION OF CONTROLS
- Positive control: The positive control chemical (cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile
- Reference Control A (verification of the HPLC system suitability): Samples containing 0.5 mM peptide and were dissolved in the appropriate peptide buffer and acetonitrile, n=1 with 3 fold injection
- Reference Control (stability of the reference controls over time): Samples containing 0.5 mM peptide were dissolved in the appropriate peptide buffer and acetonitrile, n=6
- Reference Control C1 (peptide stability control for the solvent used to dissolve the test item and the positive control): Samples containing 0.5 mM peptide were dissolved in the appropriate peptide buffer and acetonitrile, n=3
- Reference Control C2 (peptide stability control for the solvent used to dissolve the test item): Samples containing only 0.5 mM peptide were dissolved in the appropriate peptide buffer and deionized water, n=3
- Co-elution Control: A sample was prepared of the respective peptide buffer and the test item or the positive control without peptide, n=1, each

The reference control A sample and the reference control B samples of both peptides were prepared at a concentration of 500 μM in acetonitrile. Reference control C samples were prepared at a concentration of 500 μM in deionized water (C2, the solvent used to dissolve the test item) and in acetonitrile (C1, the solvent used to dissolve the positive control).

PREPARATION OF THE TEST ITEM
The test item was dissolved immediately before testing in deionised water to prepare a 100 mM stock solution.

PREPARATION OF THE PEPTIDE STOCK SOLUTIONS
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of the appropriate peptide in approximately 20 mL of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).

PREPARATION OF PEPTIDE CALIBRATION STANDARDS
Calibration standards of both peptides were prepared in a solution of 20% acetonitrile buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide. The following calibration solutions were prepared from the peptide stock solution of each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A blank of the dilution buffer was also included in the standard calibration curve for both peptides. The blank was 25% acteonitrile buffer solution with phosphate buffer pH 7.5 for the cysteine peptide and with ammonium acetate buffer pH 10.2 for the lysine peptide without peptide.

PREPARATION OF POSITIVE CONTROL AND CYSTEINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
Triplicate solutions each of the positive control and test item stock solutions were diluted with the cysteine peptide stock solution so as to prepare solutions containing 500 μM cysteine and 5 mM of Cinnamaldehyde or 5 mM of the test item. For the co-elution control, buffer solution was used in place of the cysteine stock solution.


PREPARATION OF POSITIVE CONTROL AND LYSINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
Triplicate solutions each of the positive control and test item stock solutions were diluted with the lysine peptide stock solution to prepare solutions containing 500 μM lysine and 25 mM of Cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer solution was used in place of the lysine stock solution.

TREATMENT
500 μM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5 - 30ºC for 24 ± 2 hours prior to initiation of the analysis run. The test item and the positive control were analysed in triplicate for both peptides. The appearance of the test item and positive control samples in the HPLC vials was visually inspected and documented after preparation and prior to initiation of the HPLC run.

METHOD OF ANALYSIS
Pre-column: Security Guard C18; 4,0 x 2,0 mm ID
Column: Zorbax SB C18; 2,1 x 100 mm; 3,5 μm
Eluent A: 0.1 % trifluoroacetic acid (TFA) in water
Eluent B: 0.085 % trifluoroacetic acid (TFA) in acetonitrile (ACN)
Flow rate: 0.35 mL/min
Detector: DAD (diode-array detector)
Wave length: 220 nm und 258 nm
Oven temperature: 30 °C
Injection volume: 2 μL
Run time: 26 min

DATA EVALUATION
The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

- Calculation of peptide depletion: [1- (Peptide peak area in replicate injection/mean peptide peak area in reference control c)]x100

ACCEPTANCE CRITERIA
- Linearity of standard calibration curve: coefficient of determination (r2) > 0.99
- Mean peptide depletion value of positive control: 60.8% - 100% for the cysteine peptide, 40.2% - 69.0% for the lysine peptide
- Maximum standard deviation positive control: < 14.9 percent points for cysteine depletion, < 11.6 percent points for the lysine depletion
- Mean peptide concentration of the Reference Controls A: 0.45 - 0.55 mM
- CV of peptide peak areas of reference controls B and C1: < 15%
- Maximum SD for the test item: < 14.9 percent points for cysteine depletion, < 11.6 percent points for the lysine depletion
- Mean peptide concentration of Reference Controls C (C1 and C2): 0.45 to 0.55 mM

Key result

Run / experiment:

other: 1

Parameter:

other: Mean cysteine and lysine % depletion

Value:

1.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: mean peptide depletion (%) for lysine

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: mean peptide depletion (%) for cysteine

Value:

2.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table 3: All analytical acceptance criteria for each peptide run were met:

	Peptide	Standard Linearity	Positive control depletion (percent points)	Reference controls (mean peptide concentration / coefficient of variation)	SD Test item depletion (percent points)
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9%)	450 - 550 µM (CV <15%)	SD <14.9%
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6%)	450 - 550 µM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r^2=0.9999	70.3(SD, 0.461%,n=3)	A: 498 µM (CV 0.552%, n=1) B:  486 µM (CV 1.75%, n=6) C1:          491 µM (CV 1.92%, n=3) C2:          487 µM (CV 2.01%, n=3)	SD 2.34% (n=3)
	Lysine	r^2=0.9999	42.8 (SD, 2.96%, n=3)	A: 520 µM (CV 1.12%, n=1) B:  496 µM (CV 3.10%, n=6) C1:          502 µM (CV 0.729%, n=3) C2:          500 µM (CV 1.13%, n=3)	SD 1.13% (n=3)

 

 

Table 4: The depletion of peptide in the presence of Art. 137119 (Phospho-L-Tyrosine Di Sodium Salt) was:

	Mean peak area of reference controls	Mean peak area of peptide with test item	Mean peptide depletion (%)	Mean of cysteine and lysine (%)
Cysteine	Control B: 2977821 (n=6) Control C1: 3003039 (n=3)	2895657 (n=3)	2.78	1.95
Lysine	Control B: 3084697 (n=6) Control C1: 3124787 (n=3)	3082163 (n=3)	1.12

 

Interpretation of results:

GHS criteria not met

Conclusions:

With no to minimal mean depletion of both peptides (1.95%) in the presence of the test item, it is therefore predicted as negative and not to be a potential skin sensitiser in the DPRA.

Executive summary:

The purpose of this study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item.

This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

The test item was dissolved in deionized water when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C.

There were no co-elution peaks in either the cysteine or lysine assays.

Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.95%, based on this assay the test item is placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

WoE, DPRA (OECD guideline 442C)

The purpose of this study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in deionized water when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. There were no co-elution peaks in either the cysteine or lysine assays. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.95%, based on this assay the test item is placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitiser.

 

WoE, in vitro ARE-Nrf2 Luciferase Test Method (OECD 442D)

The test item was completely dissolved in treatment culture medium up to a concentration of 2000 µM. In the first run, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. In the second run, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. 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 data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for sensitisation under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.