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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
EC Number:
948-778-9
Molecular formula:
C14O8H19N2Na3
IUPAC Name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
Test material form:
liquid
Details on test material:
Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23ADB/50
- Expiration date of the lot/batch: 21/11/2023


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
Method in compliance with current regulatory requirements
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM from MatTek Corporation (3D system of reconstructed epidermis of normal human keratinocytes)
- Tissue batch number(s): keratinocyte strain: 00267
- Production date: 16-01-2019
- Shipping date: n/a
- Delivery date: n/a
- Date of initiation of testing: 16-01-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure time the product and the controls were removed and the tissues rinsed for several times with DPBS (up to 25 times if necessary).
- Observable damage in the tissue due to washing: n/a
- Modifications to validated SOP: n/a

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT thiazolyl blue tetrazolium 5 mg/ml (MTT-100-CON) has been diluted with MTT diluent (MTT-100-DIL) up to 1 mg/ml.
- Incubation time: 3h
- Spectrophotometer: The absorbance is measured at 570 nm by microplate reader using a GEN5 software (Biotek).

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
-The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
According to EU classification, the irritancy potential of test substances is predicted to distinguish between H315 skin irritating (category 2) and not classified test substances (EU CLP).
In this study the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance.
The test substance is considered to be irritant to skin (H315), if the mean relative viability after 60 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µl
- Concentration (if solution): n/a applied neat

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): (H2O) 30 µl
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution):SDS 5% in DPBS
Duration of treatment / exposure:
25 min at room temperature
residual incubation time to 60 minutes has been done at 37±1°C, 5% CO2.
Duration of post-treatment incubation (if applicable):
At the end of the exposure time the product and the controls were removed and the tissues rinsed for several times with DPBS (up to 25 times if necessary).
Each tissue was then transferred in 6-well plate with 1 ml of Assay Medium and incubated for 24±2 hours at 37±1°C, 5% CO2, then the tissues will be transfer in a renewed Assay Medium for 18±2 h at 37±1°C, 5% CO2 before the MTT test.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean value
Value:
73.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:

ACCEPTABILITY CRITERIA
Negative control: the mean OD570nm value of the negative control tissues should be ≥0.8 and ≤2.8.
Positive control: the mean viability value of positive control tissues, expressed as % against negative control tissues should be ≤20%.
Standard deviation (SD): the SD calculated from individual % tissue viabilities of the 3 treated replicates should be ≤18%.

Any other information on results incl. tables

RESULTS

Optical density (OD Value) at 570 nm.

 

Tissue 1

Tissue 2

Tissue 3

Replicates

1

2

3

1

2

3

1

2

3

Negative control

2,283

2,307

2,283

2,386

2,339

2,346

2,480

2,460

2,471

Positive control

0,111

0,110

0,111

0,127

0,127

0,128

0,119

0,116

0,117

Sample

1,588

1,599

1,572

1,867

1,835

1,845

1,840

1,803

1,803

 

ASSAY VALIDITY CRITERIA

Value

Acceptability

Result

Negative control

Mean OD value

2.33

0.8 and2.8

Complies

Positive control

Mean Viability %

3.28

≤ 20

Complies

SD

0.347

≤ 18

Complies

Sample

SD

6.139

Complies

 

SAMPLE

% VIABILITY

TRISODIUM (2S)-2,6-BIS(3-
CARBOXYLATOPROPANAMIDO)HEXANOATE

73.30

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
On the basis of the results, interpreted according to OECD 439 the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” must be considered NOT IRRITANT for the skin.
Executive summary:

On the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” an in

vitro toxicological study aimed to evaluate any potential cutaneous irritation was carried out.

The following test was performed:

-In vitro skin irritation test on Reconstructed Human Epidermis according to OECD N. 439:2015.

To perform the in vitro skin irritation test, three-dimensional Reconstructed Human Epidermis (RHE) tissues, consisting of normal human keratinocytes cultured for 17-days on an inert 0.63 cm2polycarbonate filter at the air-liquid interface, were used.

The test item was topically applied on three tissues replicates for 60 minutes. Exposure was followed by rinsing with phosphate buffer saline (DPBS) and dried, then tissues were transferred to fresh medium and incubated for 42 additional hours, then tissues were transferred to MTT for 3 hours. The aim of this assay was to assess quantitatively the effects of the tested product on cell survival through the MTT assay.

Cell viability determination is based on cellular dehydrogenase activity, measured by MTT reduction and conversion into blue formazan salt that is quantified after extraction from tissues.

\The percentage reduction in viability is used to predict the irritation potential.

On the basis of the results, interpreted according to OECD 439:2015 the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” must be considered NOT IRRITANT for the skin.