Sodium 2-heptyl-2,3-dihydro-3-(2-hydroxyethyl)-1H-imidazole-1-propionate

Endpoint summary

Description of key information

Eye irritation - Interpretation of Results following Prediction Model

• A test item is considered to be irritant to the eye (i.e. would require labelling as either GHS 1 or 2) if the eye model viability after exposure and post-treatment incubation is ≤60%.  

• A test item is considered as a non-irritant to the eye (i.e. would not require a warning label in the European chemical classification systems) if the eye model viability after exposure and post-treatment incubation is >60%.

The current assay is not intended to differentiate between GHS class 1 and GHS class 2 or R36 and R41       (degree of stromal damage).

The percentage of viability obtained with test item Crodateric CYAP-100 was 97.3%, therefore it has to be considered as Non- Irritant to the eye.

Skin Irritation -

The skin irritation potential of Crodateric CYAP-100 was assessed in vitro according to OECD Test Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

After a 65 minute exposure on the surface of the EpiDermTM reconstructed human epidermis and a 42 ± 4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control. The percentage of viability obtained was 4.816 % and therefore since the test item reduced the viability to below 50% it should be considered as Irritant to the skin.

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

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)

Version / remarks:

“EpiDermTM In vitro Reconstructed Human Epidermis Skin Irritation Test (OECD 439 28 Jul 2015 and is based on the MatTek protocol (MK-24-007-0023, 11 July 14) using the MatTek Corporation EpiDermTM reconstructed tissue model EPI-200

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Supplier Croda
Test Item name Crodateric CYAP-100
Supplier Code DEV080817DS3
Supplier batch/lot number PS-196-562
CAS number 70983-43-6
Purity 99.41% (from CofA)
Expiry Date 3 July 2018
Physical state Solid, Dark
Storage Conditions Room Temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Justification for test system used:

The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS).

A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

After pre-wetting tissues with 25µl DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), single topical application of nominal 25mg neat test item, 30µl of negative control (DPBS) or positive control (SDS 5%) to the surface of the EpiDermTM model for a set time.

Duration of treatment / exposure:

60 ± 1 minute* (25 minutes* at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% Relative Humidity (RH)), followed by 42 ± 4h post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3)

Duration of post-treatment incubation (if applicable):

42 ± 4h post-treatment incubation

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Percentage of viability (relative to negative control)

Value:

4.816

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Mean and SD of cell viability measurements and of viability percentages after 65 min of application and 42h (± 4h) post-incubation.

 

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
DPBS	NC	1.514	0.079	100.000	5.243	5.243	Non-Irritant
SDS 5%	PC	0.077	0.018	5.097	1.176	23.064	Irritant
Crodateric CYAP-100	TA1	0.082	0.017	5.427	1.096	20.191	Irritant

 

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA1: Crodateric CYAP-100.

 

Prediction model of irritancy: test items that reduce the viability to 50% or below are irritant (I), test items with a percentage viability above 50% are considered to be non-irritant (NI).

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The skin irritation potential of Crodateric CYAP-100 was assessed in vitro according to OECD Test Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.
After a 65 minute exposure on the surface of the EpiDermTM reconstructed human epidermis and a 42 ± 4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control. The percentage of viability obtained was 4.816 % and therefore since the test item reduced the viability to below 50% it should be considered as Irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

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)

Version / remarks:

The detailed method followed in this study is described in XCellR8 SOP L0069: ‘EpiOcularTM Eye Irritation Test (OECD TG492) and is based on the MatTek protocol (MK-24-007-0055, 10 Feb 17) using the MatTek Corporation EpiOcularTM reconstructed tissue model OCL-200. This protocol was approved as the new OECD TG492 at the 2015 meeting of the OECD Working Group of the National Coordinators for the Test Guidelines Programme

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Supplier Croda
Test Item Name Crodateric CYAP-100
Supplier Code DEV080817DS3
Supplier batch/lot number PS-196-562
CAS number 70983-43-6
Purity 99.41% (from Sponsor Certificate of Analysis)
Expiry Date 3 July 2018
Physical state Solid, Dark, Brown
Storage Conditions Room Temperature
Solubility N/A - neat application
Administration method Topical application
Concentration tested Neat
XCellR8 test item code CRO1026
Study test item code TA1

Species:

human

Strain:

other: reconstructed tissue. MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

After pre-wetting tissues with 20µl PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, single topical application of nominal 50mg of neat test item, using a pin applicator or 50µl of reference items to the surface of the EpiOcularTM model for 6h ± 15 minutes, followed by a 25 ± 2 minutes’ post-treatment immersion, and 18 hours ± 15 minutes’ post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).

Duration of treatment / exposure:

After pre-wetting tissues with 20µl PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, single topical application of nominal 50mg of neat test item, using a pin applicator or 50µl of reference items to the surface of the EpiOcularTM model for 6h ± 15 minutes, followed by a 25 ± 2 minutes’ post-treatment immersion, and 18 hours ± 15 minutes’ post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).

Details on study design:

The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.
The EpiOcular™ Eye Irritation Test (EIT), using the MatTek EpiOcularTM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcularTM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Draize Rabbit Eye Test with excellent correlation -In vivo to In vitro test results.

Irritation parameter:

other: percentage of viability

Run / experiment:

The test item Crodateric CYAP-100 did not reduce the viability to 60% or below after 6h of application and should be considered as non-Irritant to the eye.

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Prior to the study, the required compatibility checks (as per SOP L0069) show that the test item did interfere with MTT. However, OD obtained with the freeze-killed tissues were low (<0.1) and therefore they were not subtracted from OD values obtained during the main assay.

Mean and SD of viability measurements and of viability percentages after 6h (± 15 min) of application and 18h (± 15 min) post-incubation.

 

Name	Code	mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
Sterile water	NC	1.421	0.068	100.0	4.8	4.8	Non-Irritant
Methyl Acetate	PC	0.035	0.025	2.4	1.7	71.6	Irritant
CRODATERIC CYAP-100	TA1	1.383	0.098	97.3	6.9	7.1	Non-Irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

Interpretation of Results following Prediction Model
• A test item is considered to be irritant to the eye (i.e. would require labelling as either GHS 1 or 2) if the eye model viability after exposure and post-treatment incubation is ≤60%.
• A test item is considered as a non-irritant to the eye (i.e. would not require a warning label in the European chemical classification systems) if the eye model viability after exposure and post-treatment incubation is >60%.
The current assay is not intended to differentiate between GHS class 1 and GHS class 2 or R36 and R41 (degree of stromal damage).
The percentage of viability obtained with test item Crodateric CYAP-100 was 97.3%, therefore it has to be considered as Non- Irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The % viability results obtained for this substance by in-vitro eye irritancy testing gave a value of 97.3%, which is much higher than the <60% value required to classify as an irritant.Therefore this substance does not meet the GHS criteria for classification for eye irritancy.

However the % viability result for skin irritation was 4.816 % and therefore since the test item reduced the viability to well below 50% and according to this in-vitro method it does meet the GHS criteria for classification and is considered as an Irritant to the skin.