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Description of key information

Based on the finding in an in-vitro skin irritation study, the substance does not meet the criteria for classification as skin irritant. Results from an in-vitro eye irritation study indicate that the substance is not irritating for the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

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

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
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%.

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)
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


PRE-TEST
Optical properties of test item or its action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability for this reason Pre-tests have been performed to allow identification potential direct MTT reducers and/or interfering and define, if additional controls need to be used to detect and correct for potential interference from such test item.
Assessment for non-coloured test item
In 6-well plates, 50 ul of test sample has been added to 2 ml isopropanol, and incubated for 3 hours at
room temperature; at the end of incubation period a visual observation has been performed and any
change in colour has been recorded.
No colour change has been observed neither in water nor isopropanol.
Assessment of Direct Reduction by MTT
The test sample has been put in contact with MTT solution to detect non-specific reduction of MTT.
In a 6-well plate, 50 ul of test sample have been added to 1 ml of MTT solution 1.0 mg/ml and
incubated at 37±1°C, 5±1% CO2 for 180±15 minutes.
MTT solution in contact with the test substance has caused change in colour.
The test sample is presumed to have not the potential to stain the tissue, and has been considered no interacting with the MTT measurement this no additional controls need to be performed.
TEST
Pre-treatment
Before the beginning of the test, the tissues have been pre-treated with 20 ul of DPBS without Ca2+
and Mg2+ for 30±2 minutes at 37±1°C, 5±1% CO2, protected from light.
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcularTM from MatTek Corporation, is a 3D system consists of a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively, but not cornified, cells.
The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo.
Potential biological contaminants have been assessed by the system supplier.
The following contaminants have not been detected:
HIV-1 virus
Hepatitis B virus
Hepatitis C virus
Bacteria, yeast and other fungi
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a test sample have been tested neat
- Concentration (if solution): n/a
Duration of treatment / exposure:
30±2 minutes at 37±1°C, 5±1% CO2
Duration of post- treatment incubation (in vitro):
At the end of the treatment, the test sample and the controls have been removed from the tissues and rinsed with 100 ml of DPBS for 3 times without Ca2+ and Mg2+.
Each tissue have been transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature, then transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2.
Number of animals or in vitro replicates:
2 replicates, 3 repetition each
Details on study design:
- Details of the test procedure used

CULTURE MEDIA AND REAGENTS
The validity of culture media and reagent has been assessed before starting the analyses.
EpiOcularTM (MatTek Corporation)
Assay Medium (OCL-200-ASY) (MatTek Corporation)
Dulbecco’s Phosphate buffer solution (DPBS) (MatTek Corporation)
MTT thiazolyl blue tetrazolium (MTT-100-CON) (MatTek Corporation)
MTT diluent (MTT-100-DIL) (MatTek Corporation)
Isopropyl alcohol (IPA) (MTT-100-EXT) (MatTek Corporation)
Methyl Acetate (MatTek Corporation )
Ultrapure Water (Eurospital))

SOLUTIONS
MTT SOLUTION: MTT thiazolyl blue tetrazolium 5 mg/ml has been diluted with MTT diluent up to 1 mg/ml.
The MTT SOLUTION has been prepared at use, sheltered from light, and discarded at the end of the study.

EQUIPMENT
The validity of instruments and equipment has been assessed before starting the analyses.
Laminar flow filtered work area (Flow)
CO2 incubator (Flow)
Chronometer (Oregon Scientific)
Microplate reader Mod EL800 (Bio-Tek)

- RhCE tissue construct used, including batch number :
EpiOcularTM from MatTek Corporation, is a 3D system consists of a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively, but not cornified, cells.
The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo.

- Doses of test chemical and control substances used :
Assay Sample
50 µl of the test sample have been tested neat.
Controls
Positive control: 50 µl of neat Methyl Acetate have been used as positive control.
Negative control: 50 µl of ultrapure water have been used as negative control.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Treatment
The test sample, the negative and positive control have been topically applied on tissues replicates for 30±2 minutes at 37±1°C, 5±1% CO2, to respect the exposure times the applications have been performed at intervals of not less than 1 minute.
Post treatment incubation
At the end of the treatment, the test sample and the controls have been removed from the tissues and rinsed with 100 ml of DPBS for 3 times without Ca2+ and Mg2+.
Each tissue have been transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature, then transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2.

MTT TEST
-After the post treatment, the tissues have been treated for 180±10 minutes at 37±1°C, 5±1% CO2 with 300 µl of MTT SOLUTION 1 mg/ml and then submerging in 2 ml of Isopropanol for 2 hours at room temperature with gentle agitation (about 120 rpm), for formazan extraction.
A 96-well plate has been prepared by transferring 3 replicates of 200 ul of extracts from positive control, negative control and sample, to read the optical density (OD) at 570 nm at the microplate reader. Isopropanol has been used as blank.
Optical density measurements
The absorbance is measured on a microplate reader using a 570 nm wavelength using a GEN5
software (Biotek).

ACCEPTABILITY CRITERIA
Negative control: mean OD570nm of the tissue of negative control should be >0.8 and <2.5 .
Positive control: mean viability of the tissue replicates exposed for 30 min with the positive control, expressed as % of the negative control, should be <50%.
Difference of viability: the difference of viability between two tissue replicates should be <20%.
INTERPRETATION OF RESULTS
The OD values obtained with the replicate tissue extracts is used to calculate the mean percent tissue viability normalised to the negative control, which is set at 100%.
The percentage tissue viability cut-off value for identifying test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) is given in Table below for Prediction Models according to UN GHS classification.
Results should thus be interpreted as follows:
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) the established percentage tissue viability cut-off value, as shown in Table for Prediction Models according to UN GHS classification
In this case no further testing in other test methods i s required.
- If the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to the established percentage tissue viability cut-off value, no prediction can be made, as shown in Table for Prediction Models according to UN GHS classification.
Table: Prediction Models according to UN GHS classification
Tissue No Category No prediction can be made
EpiOcularTM EIT Mean tissue viability>60% Mean tissue viability≤60%
Irritation parameter:
other: viability vs negative control
Value:
87.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the mean percent tissue viability after exposure and post-exposure incubation is >60%, the established percentage tissue viability cut-off value, for Prediction Models according to UN GHS classification

RESULTS

Optical Density
Measurements OD Value
570 nm

Tissue 1

Tissue 2

Repetition

1

Repetition

2

Repetition

3

Repetition

1

Repetition

2

Repetition

3

Blanks

0,041

0,041

0,041

0,041

0,041

0,041

Negative control (NC)

2,464

2,487

2,435

2,508

2,479

2,459

Positive control (PC)

0,087

0,086

0,088

0,075

0,075

0,074

Test sample

2,127

2,114

2,089

2,222

2,232

2,224

 

ASSAY VALIDITY CRITERIA

Value

Acceptability

Result

Negative
control

Mean OD value

2.431

0.8 and2.5

Comply

Difference of viability %

0.822

<20%

Comply

Positive
control

Mean % Viability

1.65

<50%

Comply

Difference of viability %

0.493

<20%

Comply

Sample

Difference of viability %

4.772

<20%

Comply

 

SAMPLE

% VIABILITY

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

87.5

Interpretation of results:
GHS criteria not met
Conclusions:
On the basis of the results, interpreted according to OECD 492:2017, can be stated that the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” belong to No Category for eye.
Executive summary:

On the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” anin vitro toxicological study aimed to evaluate ocular irritation potential has been carried out.

The following test has been performed:

-in vitro ocular irritation – Reconstructed EpiocularTMtissue model test method.

To perform thein vitro Ocular Irritation on EpiOcularTM, a Tissue Model from MatTek Corporation, Cornea-Like 3-D Tissue Structure, has been used.

MatTek’s EpiOcular system consists of normal, primary human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure which closely parallels the corneal epithelium.

Histological examination of the RhCE tissue construct on one tissue not treated that should demonstrate appropriated human cornea-like epithelium structure (including at least 3 layers of viable epithelial cells and a non-keratinized surface) has been performed.

Pre-tests to identify identification potential direct MTT reducers and/or interfering and define, if additional controls need to be used to detect and correct for potential interference from such test item, have been performed.

On the basis of obtained results in pre-tests, no additional controls have been performed.

To performin vitro Ocular Irritation test, two replicates of three tissues series, one for test sample, one for negative control and one for positive control have been used.

The test sample has been topically applied on tissues replicates for 30 minutes at 37±1°C, 5±1% CO2. At the end of the treatment tissues have been rinsed with 100 ml for 3 times of DPBS without Ca2+and Mg2+.then transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature in order to remove any test article absorbed by the tissues, then tissues have been transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2.

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 aim of this assay has been to assess quantitatively the effects of the tested product on cell survival through the MTT assay.

After incubation, tissues have been treated with MTT SOLUTION 1 mg/ml for 180±10 minutes at 37±1°C, 5±1%CO2,then tissues have been treated with Isopropanol for 2 hours at room temperature with gentle agitation (about 120 rpm) for formazan extraction. The percentage reduction in viability is used to predict the irritation potential.

On the basis of the results, interpreted according to OECD 492:2017, can be stated that the test item TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE belong to No Category for eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance was tested for skin and eye irritation in two in-vitro studies.

Based on the findings in these studies, the substance is NOT classified as a skin irritant nor as eye irritant according to CLP (Regulation EC No. 1272/2008).

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