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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

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.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

860-352-3

Cas Number:

1610350-91-8

Molecular formula:

C9H10NO6PNa2

IUPAC Name:

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

Test material form:

solid: particulate/powder

In vitro test system

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.

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Results 7

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

Results 8

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.