Sodium hydrogen L-aspartate

Endpoint summary

Description of key information

Skin sensitisation:

In an in vitro direct peptide reactivity assay (DPBA) according to OECD Guideline 442C, the test substance showed a cystein depletion of 0.57 % and a lysine depletion of 0.42 %, indicating no sensitising properties (reference 7.4.1 -1).

In an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D, the test substance did not induce luciferase activity (reference 7.4.1 -2).

In conclusion, the test substance is considered to be a non-sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-11 to 2017-04-22

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))

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design: The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides.
The mean peptide depletion of the positive control for the cysteine peptide was between 60.8 % and 100 % (74.90 %).
The mean peptide depletion of the positive control for the lysine peptide was between 40.2 % and 69.0 % (54.93 %).

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0.42

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD7	0.0000	0.0000	0.0000	0.0000
STD6	153.8068	0.0167	139.6547	0.0167
STD5	321.6256	0.0334	281.4845	0.0334
STD4	642.2243	0.0667	555.3300	0.0667
STD3	1318.5444	0.1335	1100.1805	0.1335
STD2	2593.4377	0.2670	2176.1533	0.2670
STD1	5075.1016	0.5340	4280.0898	0.5340

 

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1193.1283	0.1239	74.99	74.90	0.25	0.34
	1187.7434	0.1233	75.10
	1210.7732	0.1258	74.62
Test Item	4777.8320	0.5002	0.00*	0.57	0.52	91.06
	4674.7964	0.4893	1.01
	4689.8892	0.4909	0.69

* value set to zero due to negative depletion

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1861.8433	0.2304	55.78	54.93	0.78	1.42
	1926.2322	0.2384	54.25
	1904.2300	0.2357	54.77
Test Item	4143.4482	0.5151	0.80	0.27	0.46	173.21
	4257.1900	0.5293	0.00*
	4338.8200	0.5395	0.00*

* values set to zero due to negative depletion

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model¹

Mean Cysteine andLysine PPD	ReactivityClass	DPRA Prediction²
0.00 % ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38 % < PPD ≤ 22.62%	Low Reactivity	Positive
22.62 % < PPD ≤ 42.47%	Moderate Reactivity
42.47 % < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 prediction model¹

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89 %	No orMinimalReactivity	Negative
13.89% < PPD ≤ 23.09 %	LowReactivity	Positive
23.09% < PPD ≤ 98.24 %	ModerateReactivity
98.24% < PPD ≤ 100 %	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of anIATA.

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.42	Minimal Reactivity	no sensitizer	0.57	Minimal Reactivity	no sensitizer
Positive Control	64.92	High Reactivity	sensitizer	74.90	Moderate Reactivity	sensitizer

 

Interpretation of results:

other: In this study the test item showed minimal reactivity towards the peptides. The test item can be classified as “non-sensitiser”.

Remarks:

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.

Conclusions:

In an in vitro direct peptide reactivity assay (DPBA) according to OECD Guideline 442C, the test substance showed a cystein depletion of 0.57 % and a lysine depletion of 0.42 %, indicating no sensitising properties.

Executive summary:

In an in vitro direct peptide reactivity assay according to OECD Guideline 442C, the sensitising potential of L-Aspartic acid sodium salt monohydrate was determined (reference 7.4.1 -1) The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any sample.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both peptides by comparing the peptide concentration of the test item samples to the corresponding reference control C.

In the lysine run a shift of the lysine peptide peak from 7.7 min to 7.0 up to 6.5 min was observed. Since the reference control samples allow clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, that indeed the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. Furthermore, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the results. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38 % (0.42 %). Based on the prediction model 1 the test item can be considered as non-sensitiser. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92 %.

Based on these results, the test substance was considered as non-sensitising.

Furthermore, it was assumed, that the anhydrate form has the same sensitising potential and thus the result of the test item is also applicable.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-05 to 2017-05-24

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)

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
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.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.28 in experiment 1; 2.65 in experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

98.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.41

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

77.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	119.3	108.4	113.9	7.7
	8.00	105.9	120.5	113.2	10.3
	16.00	112.8	120.6	116.7	5.5
	32.00	121.9	126.1	124.0	3.0
	64.00	128.7	133.3	131.0	3.2
Test Item	0.98	95.3	81.1	88.2	10.0
	1.95	98.3	77.6	87.9	14.7
	3.91	91.9	85.7	88.8	4.4
	7.81	96.3	89.0	92.6	5.1
	15.63	95.5	89.4	92.4	4.3
	31.25	90.1	88.5	89.3	1.1
	62.50	95.4	86.9	91.1	6.0
	125.00	89.9	86.2	88.1	2.6
	250.00	85.5	85.9	85.7	0.3
	500.00	91.7	85.5	88.6	4.3
	1000.00	86.4	81.8	84.1	3.2
	2000.00	91.4	77.4	84.4	9.9

 

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	1.05	1.50	1.19	1.25	0.23
	8.00	1.38	1.20	1.30	1.29	0.09
	16.00	1.33	1.21	1.27	1.27	0.06	*
	32.00	1.43	1.52	1.32	1.42	0.10
	64.00	4.85	3.80	4.18	4.28	0.53	*
Test Item	0.98	1.03	0.70	1.06	0.93	0.20
	1.95	0.90	0.66	1.43	1.00	0.39
	3.91	1.12	0.78	1.08	0.99	0.19
	7.81	0.85	0.66	1.20	0.90	0.27
	15.63	0.83	0.67	0.92	0.81	0.13
	31.25	0.91	0.62	0.89	0.80	0.16
	62.50	0.75	0.61	0.81	0.72	0.10
	125.00	0.94	0.62	1.05	0.87	0.22
	250.00	0.80	0.56	0.83	0.73	0.14
	500.00	0.88	0.58	0.93	0.79	0.19
	1000.00	0.71	0.52	0.91	0.71	0.20
	2000.00	0.89	0.71	0.92	0.84	0.12

* = 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	1.12	1.19	1.15	1.15	0.04
	8.00	1.10	1.24	1.27	1.21	0.09
	16.00	1.75	1.33	1.53	1.54	0.21	*
	32.00	1.87	1.70	1.96	1.85	0.13	*
	64.00	2.31	2.72	2.92	2.65	0.31	*
Test Item	0.98	1.25	1.20	1.60	1.35	0.22
	1.95	1.35	1.31	1.31	1.32	0.02
	3.91	1.25	1.10	1.28	1.21	0.10
	7.81	1.08	1.05	1.17	1.10	0.06
	15.63	1.13	1.15	1.30	1.19	0.09
	31.25	1.24	1.08	1.18	1.17	0.08
	62.50	1.28	1.19	1.33	1.27	0.07
	125.00	1.07	1.14	1.07	1.09	0.04
	250.00	1.21	1.21	1.30	1.24	0.05
	500.00	1.06	1.15	1.17	1.13	0.06
	1000.00	1.42	1.30	1.45	1.39	0.08
	2000.00	1.33	1.45	1.43	1.41	0.06

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

Table 4: Induction of Luciferase Activity – 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	1.25	1.15	1.20	0.06
	8.00	1.29	1.21	1.25	0.06
	16.00	1.27	1.54	1.40	0.19
	32.00	1.42	1.85	1.63	0.30
	64.00	4.28	2.65	3.46	1.15
Test Item	0.98	0.93	1.35	1.14	0.30
	1.95	1.00	1.32	1.16	0.23
	3.91	0.99	1.21	1.10	0.15
	7.81	0.90	1.10	1.00	0.14
	15.63	0.81	1.19	1.00	0.27
	31.25	0.80	1.17	0.99	0.26
	62.50	0.72	1.27	0.99	0.39
	125.00	0.87	1.09	0.98	0.16
	250.00	0.73	1.24	0.98	0.36
	500.00	0.79	1.13	0.96	0.23
	1000.00	0.71	1.39	1.05	0.48
	2000.00	0.84	1.41	1.12	0.40

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

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	-	-
Imax	1.00	1.41	1.20	0.29
IC30[µM]	n.a.	n.a.	-	-
IC50[µM]	n.a.	n.a.	-	-

n.a.= not applicable

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	19.7	pass	12.5	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	1.0	pass	3.0	pass
EC1.5PC	7 < x < 34 µM	32.87	pass	15.08	pass
Induction PC at 64 µM	2.00 < x < 8.00	4.28	pass	2.65	pass

 

Interpretation of results:

other: Under the condition of this study the test substance did not induce the luciferase activity and is threrefore considered as non sensitiser.

Remarks:

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.

Conclusions:

In an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D, the test substance did not induce luciferase activity.

Executive summary:

In the present study, the sensitising properties of L-Aspartic acid sodium salt monohydrate was determined in an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D.

Based on a molecular weight of 173.10 g/mol a stock solution of 200 mM was prepared in water. 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 (I_max) induction of 1.00 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 98.3 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, no cytotoxic effect was observed. In the second experiment, a max luciferase activity (I_max) induction of 1.41 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 77.4 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. Furthermore, no cytotoxic effect was observed. 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.

Furthermore, it was assumed, that the anhydrate form has the same sensitising potential and thus the result of the test item is also applicable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

According to REACH the skin sensitisation study in testing animals is replaced by in vitro methods. Up to now, no single in vitro test can replace the animal study by its own. Therefore an integrated testing strategy is necessary to assess the sensitising potential of the test substance in vitro. The testing strategy consists of the direct peptide reactivity assay (DPRA) and the ARE-Nrf2 Luciferase assay, representing key elements in the skin sensitisation process.

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the skin sensitising potential of L-Aspartic acid sodium salt monohydrate was determined (reference 7.4.1 -1). The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any sample.

No co-elution of test item with the peptide peaks was observed. Sensitizingpotential of the test item was predicted from the mean peptide depletion of both peptides by comparing the peptide concentration of the test item samples to the corresponding reference control C.

In the lysine run a shift of the lysine peptide peak from 7.7 min to 7.0 up to 6.5 min was observed. Since the reference control samples allow clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, that indeed the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. Furthermore, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the results. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38 % (0.42 %). Based on the prediction model 1 the test item can be considered as non-sensitiser. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92 %. Based on these results, the test substance was considered as non-sensitising.

Since the DPRA can not exclude a skin sensitising potential of the test substance on its own, the skin sensitising property of L-Aspartic acid sodium salt monohydrate was determined in an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D (reference 7.4.1 -2). Based on a molecular weight of 173.10 g/mol a stock solution of 200 mM was prepared in water. 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 (I_max) induction of 1.00 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 98.3 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated. Furthermore, no cytotoxic effect was observed. In the second experiment, a max luciferase activity (I_max) induction of 1.41 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 77.4 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated. Furthermore, no cytotoxic effect was observed. 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.

In conclusion, both assays of the integrated testing strategy did not show a skin sensitising potential of the test substance. Therefore the test substance is considered as non-sensitiser. Furthermore, it was assumed, that the anhydrate form has the same sensitising potential and thus the result of the test item is also applicable.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.