L-aspartic acid, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 june 2020

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-amino-3-carboxypropanoate

Cas Number:

323194-76-9

Molecular formula:

C4H7NO4.xNa

IUPAC Name:

sodium 2-amino-3-carboxypropanoate

Test material form:

solid: particulate/powder

Details on test material:

Name: L-aspartic acid, sodium salt monohydrate 1
Batch/Lot number: Z201125/ VG29848563
CAS number: 323194-76-9
Anhydrous substance name: L-aspartic acid, sodium salt, CAS: 17090-93-6
Appearance: Solid, white, crystalline powder
Purity: 98%
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed
container)

Specific details on test material used for the study:

Name: L-aspartic acid, sodium salt monohydrate
CAS number: 323194-76-9
Lot number: Z201125/ VG29848563
Description: Crystalline solid, white powder
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

In vitro test system

Test system:

human skin model

Source species:

other: EpiDerm (Source: MatTek Corporation, USA) is a three-dimensional human epidermis model.

Cell type:

non-transformed keratinocytes

Cell source:

other: Source: MatTek Corporation, USA

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm (Source: MatTek Corporation, USA) is a three-dimensional human epidermis model. It consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation. Ultrastructurally, the EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology [5].

The EpiDerm Skin Model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. EpiDerm consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. EpiDerm is mitotically and metabolically active. Markers of mature epidermis specific differentiation such as profilaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase have been localized in the model. Ultrastructural analysis has revealed the presence of keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis [

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test item was applied on the surface of the tissues with 1 minute interval between two applications, under laminar flow hood.

Duration of treatment / exposure:

Following the end of the last application, all plates were placed into the incubator (37±1°C, 5±1% CO2, 90±10% RH) for 35±1 minutes.

Duration of post-treatment incubation (if applicable):

After 35 minutes, all plates were removed from the incubator, and kept at room temperature under the sterile hood until the period of 60-minutes exposure period had been completed for the first dosed tissue.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

>= 88.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

  VIABILITY RESULTS

 

The results of the Optical Density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2. The mean OD value for the test item treated skin tissues showed 88.6% of the Negative Control (NC). NC values were not below the historically established boundaries; tissues treated with the NC item reflected their ability to respond to an irritant chemical under the conditions of this study, therefore all validity criteria met according to OECD 439.

 

Table 2 – Viability results

Tissue treated	Optical density		Viability%
	Tissue number	OD mean (corrected)	Individual	Mean
Negative control (DPBS)	#1	1.784	104.6	100
	#2	1.685	98.7
	#3	1.650	96.7
	Historical control range	1.338-1.812	–	–
Positive control (5% SDS)	#1	0.068	4.0	3.6
	#2	0.071	4.2
	#3	0.044	2.5
	Historical control range	0.006-0.160	–	–
L-aspartic acid, sodium salt monohydrate	#1	1.448	84.8	88.6
	#2	1.572	92.1
	#3	1.515	88.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item, the mean cell viability was 88.6% after 60-minute exposure, compared to the concurrent negative control. This is above the thresholds of 50%, therefore the test item was considered as being Non-irritant according to the UN GHS/EU CLP Classification criteria. The experiment met all the validity criteria; therefore, the study was considered to be valid.

In conclusion, based on the result of this present study, the test item L-aspartic acid, sodium salt monohydrate is considered not irritant to skin.

 

Executive summary:

An in vitro skin irritation test with the test item L-aspartic acid, sodium salt monohydrate was performed in a reconstructed human epidermis model. EpiDerm™ is designed to predict and classify the irritancy potential of chemicals by measuring its cytotoxic effect as reflected in the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritability of the test item was evaluated according to the OECD No. 439 guideline.

 

Three tissues per exposure time were treated for 60 minutes. Exposure of test material was terminated by rinsing with DPBS solution. The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using an extractant (isopropanol) and then quantified spectrophotometrically at 570±10 nm.

 

DPBS and aqueous SDS solution treated tissues were used as negative and positive controls, respectively (three tissues/compound). For each treated and control tissue, cell viability was expressed as a % relative to the concurrent negative control.

 

Following exposure with the test item, the mean cell viability was 88.6% after 60-minute exposure, compared to the concurrent negative control. This is above the threshold of 50%, therefore the test item was considered as being Non-irritant according to the UN GHS/EU CLP Classification. The experiment met all the validity criteria; therefore, the study was considered to be valid.

 

In conclusion, based on the result of this present study, the test item L-aspartic acid, sodium salt monohydrate is considered not irritant to skin.