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

Endpoint summary

Description of key information

Two in-vitro test studies were conducted on this substance for skin sensitisation, and both found the substance to be non-sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

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:

other: OECD Test Guideline 442E: human Cell Line Activation Test (h-CLAT)

Version / remarks:

The study was performed using the h-CLAT method as detailed in XCellR8 SOP L0094 which complies with OECD TG 442E and EURL ECVAM DB-ALM Protocol No. 158.

Principles of method if other than guideline:

To determine the In Vitro skin sensitisation potential of Crodateric CYAP-100 using the human Cell Line Activation Test (h-CLAT) method as detailed in OECD TG 442E (adopted 29 Jul 2016) and also in EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015).

GLP compliance:

yes (incl. QA statement)

Type of study:

other: human Cell Line Activation Test (h-CLAT)

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
Solubility Ethanol, Isopropanol
XCellR8 Test Item Code CRO1026
Study Test Item Code TA1

Details on the study design:

Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

Positive control results:

Several criteria were not considered acceptable when assessing Runs 1 and 2. The responses were considered to be negative for sensitisation however the viability at the top concentration (348.84 µg/ml) was not less than 90%. In addition, the positive control in Run 2 did not produce an upregulated CD54 signal. Therefore, these runs were repeated using a higher dose in order to yield a cytotoxic response at the top concentration (552 µg/ml top concentration).
In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%. This was achieved and the control was considered to be a Pass.

Key result

Parameter:

other: The dose that gave 75% cell viability in µg/ml.

Value:

290.7

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, the skin sensitisation potential of Crodateric CYAP-100 was assessed using the in vitro human Cell Line Activation Test (h-CLAT) method according to OECD Test Guideline 442E. After a 24h incubation with the test item the expression of cell surface markers CD54 and CD86 on THP-1 cells was measured by flow cytometry.
For Crodateric CYAP-100 the dose that yielded 75% cell viability was found to be 290.7 µg/ml. CD54 and CD86 expression were not upregulated above the sensitisation threshold by the test item at concentrations where the cell viability was ≥ 50% and therefore, Crodateric CYAP-100 was classified as a Non-Sensitiser as per the prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

The detailed method followed in this study is described in XCellR8 SOP L0057 ‘Skin Sensitisation Key Event 2 (KeratinoSensTM) following guideline OECD TG442D’, and is based on the KeratinoSensTM assay, adapted to fully animal-free by XCellR8 and validated in-house (studies 14XC004, 15XC001, 16XCO04) using the 10 proficiency chemicals and 11 reference chemicals described in TG 442D. Luciferase measurements and MTT viability testing were performed at the end of the appropriate exposure period (48h).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Supplier Sponsor
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 the certificate of analysis provided by the Sponsor)
Expiry Date 3 July 2018
Physical state Solid, Dark
Storage Conditions Room Temperature
Solvent 1% Ethanol in cell culture medium
Administration method In cell culture medium
Concentrations tested (µg/ml) 5000, 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, 19.5, 9.8, 4.9, 2.4
XCellR8 test item code CRO1026
Study test item code TA3

Positive control results:

Test results are acceptable if:
•The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
•The lmax and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
o average induction in the three replicates for cinnamic aldehyde at 32 µM is between the XCellR8 historical range (currently 1.6 and 3)
o EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
•CV% of blank values < 20%

Key result

Parameter:

other: EC1.5 values

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold

Other effects / acceptance of results:

Test items are considered as sensitiser if the following conditions are met in 2 of 3 repetitions:
•the Imax is higher than 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);
•the cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitizing skin irritants;
•the EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW);
•there is an apparent overall dose-response for luciferase induction (or a biphasic response);

 

	Test item concentration (µg/ml)
	2.441	4.883	9.766	19.531	39.063	78.125	156.250	312.500	625	1250	2500	5000
Mean of fold induction	0.804	0.906	0.971	0.824	0.719	0.398	-0.001	-0.003	-0.004	-0.003	-0.003	-0.003
SD	0.076	0.106	0.163	0.068	0.105	0.064	0.006	0.004	0.005	0.005	0.005	0.005
Viability %	111.73	108.13	104.22	103.04	88.30	89.08	-10.34	-11.90	-10.31	-7.28	-1.26	0.91
Imax	0.971 at 9.766µg/ml
EC1.5	No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold
IC50	109 µg/ml
IC30	93 µg/ml

Values in red are below the 70% viability threshold

 

Determination criteria for the skin sensitisation potential ofCrodateric CYAP-100
	REP1	REP2	REP3
Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:	No	No	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	N/A	N/A	N/A
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)	N/A	N/A	N/A
Does the test item induce the luciferase in a dose-dependent manner	N/A	N/A	N/A
Classification	Non-sensitiser	Non-sensitiser	Non-sensitiser

 

Interpretation of results:

GHS criteria not met

Conclusions:

The human skin sensitisation potential of Crodateric CYAP-100 was assessed using validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values.
In this study, Crodateric CYAP-100 was classified as a non-sensitiser to human skin

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)