Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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

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 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)

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


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.

Test animals / tissue source

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

Test system

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%

Results and discussion

In vitro

Results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.