Sodium hydrogen L-aspartate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen L-aspartate

EC Number:

223-264-0

EC Name:

Sodium hydrogen L-aspartate

Cas Number:

3792-50-5

Molecular formula:

C4H7NO4.Na

IUPAC Name:

sodium hydrogen 2-aminosuccinate

Test material form:

solid

In chemico test system

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.

Results and discussion

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 %).

In vitro / in chemico

Results

Results 1

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

Results 2

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.