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Diss Factsheets

Administrative data

Endpoint:
additional toxicological information
Remarks:
EpiIntestinal Small Intestine Tissue Model was used to compare the registered molecule (target), the source and other acylglutamates that have similar structure in order to better justify Read Across use.
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 21 June 2021 to 25 June 2021
Reliability:
1 (reliable without restriction)
Justification for type of information:
The test was used to evaluate toxicological behaviuor of registered substance (target) against the source substance and different acylglutamates with same structure but different fatty chain. No significative differences could be found. This helps Read Across Justification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Type of study / information:
In vitro study

Test material

Constituent 1
Reference substance name:
Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt
Cas Number:
167888-81-5
Molecular formula:
Not applicable, see single constituents
IUPAC Name:
Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt
Test material form:
liquid
Details on test material:
The product identified with the Registration Number 01-2120781106-56-0000 is always been manufactured in the same conditions with same raw materials (quality and quantity) and synthesis reaction.
So all tests done before ECHA discussion about name and identificative are still with the old name but they refer in any case to the registered molecule. The new name was changed after ECHA contact in order to find the most proper identificative for the molecule.
Specific details on test material used for the study:
Batch no.: 2268 supplied by the sponsor (also called PROTELAN AG8-EC)
33.7% in water as active ingredient (water solution), purity 100%
Solubility: soluble in water
Storage condition of test material: room temperature (15 °C - 25 °C)
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
pH (20 °C) = 10.2
More information can be found on the attached report

Results and discussion

Any other information on results incl. tables

Methods are adapted from MatTekprotocol MK-24-007-0083


 


Preliminary test             Test item checked for MTT interference


Day 1:                          Tissue receipt and overnight conditioning 


Day 2:                          Exposure to and removal of test item that has been diluted to a betaine content of 29.6% and pH adjusted to a pH of 7 ±0.2; and reference items (see table)
MTT viability test


Day 3:                          Absorbance optical density (OD) read at 570 nm.
                                   OD values exported to the analysis spreadsheet


 


 
































Item



Number of tissues



Amount applied per tissue



How applied



Exposure



Test Item



3



Single dose of 50µldiluted and pH adjusted test item 



To the surface of the EpiIntestinalTM tissue model.



15, 60 and 120 minutes



PC



3



Single dose of 100µl PC



15, 60 and 120 minutes



NC



3



Single dose of 100µl NC



60 minutes



 


After the exposure period indicated above all test chemicalswere removed by rinsing with DPBS.


Results from the MTT assay were entered into a bespoke Microsoft Excel workbook containing formulae to convert the raw results to a percentage viability for the test item relative to the negative control.  From this, an ET50 value (the effective time at which the test item caused a 50% reduction in tissue viability) and dose response curve isgenerated (data allowing).


The assay was valid.


 





















Acceptance criterion



Result



Outcome



The ET50 of the positive control should bebetween 15 and 55 minutes.



29.35mins



Pass



SD of viability for all test items and controls must be <18%.



The highest SD of viability was for the PC at 15 minutes: 11.3%



Pass*



* This is after the removal of one value from the PC at 60 minutes (tissue 2).


12Results


 


12.1  Preliminary test


 


The preliminary test confirmed that none of the four items tested interfered with MTT.


 


12.1 Viability


 


The percentage viability value for the EpiIntestinalTM models exposed to the four itemsrelative to the negative control set to 100% were calculated.


 


No outlierswereidentified.


 


 















































































































































































































































































































































































































Condition



Tissue



Raw data
(blankcorrected)



OD Mean



Viability (%)



Mean (%)



SD (%)



NC 60 min



1



1.836



1.896



1.866



103.7



100.0



3.6



2



1.773



1.814



1.793



99.7



3



1.723



1.752



1.737



96.6



PC 15 min



1



1.362



1.385



1.373



76.3



71.4



11.3



2



1.048



1.055



1.051



58.4



3



1.437



1.419



1.428



79.4



PC 60 min



1



0.615



0.636



0.625



34.8



27.2



10.6



2



1.154



1.217



1.185



65.9



3



0.351



0.359



0.355



19.7



PC 120 min



1



0.091



0.097



0.094



5.2



5.0



0.8



2



0.099



0.106



0.102



5.7



3



0.075



0.072



0.073



4.1



TA1 15 min



1



0.164



0.171



0.167



9.3



9.3



0.4



2



0.175



0.175



0.175



9.7



3



0.156



0.162



0.159



8.8



TA1 60 min



1



0.135



0.138



0.136



7.6



8.3



0.7



2



0.151



0.151



0.151



8.4



3



0.160



0.160



0.160



8.9



TA1 120 min



1



0.098



0.098



0.098



5.4



7.2



1.6



2



0.155



0.151



0.153



8.5



3



0.140



0.140



0.140



7.8



TA2 15 min



1



0.116



0.122



0.119



6.6



5.2


 



1.2


 



2



0.074



0.078



0.076



4.2



3



0.086



0.093



0.089



4.9



TA2 60 min



1



0.083



0.083



0.083



4.6



5.5



1.0



2



0.123



0.115



0.119



6.6



3



0.098



0.098



0.098



5.4



TA2 120 min



1



0.081



0.081



0.081



4.5



4.2



0.3



2



0.078



0.075



0.076



4.2



3



0.069



0.073



0.071



3.9



TA3 15 min



1



0.741



0.738



0.740



41.1



43.7



2.3



2



0.817



0.785



0.801



44.5



3



0.816



0.817



0.817



45.4



TA3 60 min



1



0.436



0.433



0.435



24.2



25.3



4.7



2



0.383



0.380



0.382



21.2



3



0.556



0.537



0.547



30.4



TA3 120 min



1



0.413



0.409



0.411



22.9



24.1



2.5



2



0.400



0.405



0.403



22.4



3



0.487



0.483



0.485



27.0



TA4 15 min



1



0.071



0.070



0.071



3.9



3.8



0.1



2



0.063



0.073



0.068



3.8



3



0.061



0.073



0.067



3.7



TA4 60 min



1



0.076



0.083



0.080



4.4



4.7



0.3



2



0.091



0.089



0.090



5.0



3



0.080



0.083



0.082



4.5



TA4 120 min



1



0.083



0.069



0.076



4.2



4.0



0.2



2



0.072



0.070



0.071



4.0



3



0.067



0.069



0.068



3.8



*orange value : excludedfrom the data processing. Whenremoved the SD% wasbelow 18%.


 





    •  




12.2ET50Determination


 


After normalisation to the negative control, percentage viability values were plotted against time (mins) on a semi-log graph.

Applicant's summary and conclusion

Conclusions:
The ET50was not able to be calculated for any of the fours items (target, source and other two acylglutamates) tested in the study 21ZSZ01EIN because the viability (%) for all timepoints recorded and was below 50% and therefore the ET50 value was subsequently classified as being <15 minutes
The PC met its required acceptance criteria and had a calculated ET50value of 29.35 minutes. No significative differences could be found among the 4 molecules
Executive summary:

The epithelial lining of the gastrointestinal (GI) tract is a gatekeeper for entry of orally ingested nutrients and xenobiotics including medicaments. The small intestine has a well organized structure containing proliferative cells which migrate along the crypt-villi axis and differentiate into functionally mature epithelial cells. To enable in vitro study of the small intestinal epithelium, MatTek has developed Epilntestinal, a model of the human small intestine.


Epilntestinal is a 3D reconstructed tissue model produced from normal, human-derived small intestine epithelial and endothelial cells and fibroblasts (EPilntestinalFT). The highly differentiated tissue model is produced at the air-liquid-interface (ALI) in easy-to-handle tissue culture inserts using serum-free medium. Structural analysis of the tissue model demonstrates columnar shaped basal cells and Kerckring folds. Ultrastructurally, Epilntestinal exhibits brush borders, functional tight junctions and mucous secreting granules mimic the in vivo tissue.


The study will be performed using the EpiIntestinalTM reconstructed tissue model SMI-100 (MatTek Corporation) using a method based on the MatTekEpiIntestinal™ Small Intestine Tissue Model (SMI-100) Use Protocol and MTT Tissue Viability (ET-50) (MK-24-007-0083 issued 02/23/2016).


 


This in vitro assessment of small intestine tissues viability is based on the effective time at which four test items caused a 50% reduction in tissue viability (ET50).