Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

Endpoint summary

Description of key information

Based on the in vitro and HRIPT study results, the test substance is considered to be non-sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 31, 2017 to February 05, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol No. 154 (Direct Peptide Reactivity Assay for Skin Sensitisation Testing)

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Details on the study design:

See below 'Any other information for materials and methods incl. tables' for details on study design.

Test and reference substances

Test substance
Test substance: Incromectant AMEA 100-LQ-MH
Supplier Code: HH40210
Supplier Batch/Lot Number: 0000974911
CAS Number: 142-26-7
Purity: 100%
Molecular Weight: 103.12
Expiry Date: 4th March 2018 (Re-test date from CofA)
Physical State: Light Amber liquid
Storage Condition: Room Temp.
Solubility: Water, Ethanol.
Solvent used: Isopropanol
Concentration tested: 100mM


Reference Substance
Supplier: Sigma-Aldrich
Reference Substance Name: Cinnamic Aldehyde
Lot Number: MKBV4784V
CAS Number: 104-55-2
Purity: >95%
Concentration Tested: 100mM
Solvent: HPLC Grade Acetonitrile (CAS No. 75-05-8)
Expiry Date: Apr 2020
Storage Conditions: Positive control is prepared fresh on Day 1 of main test from stock chemical stored at room temperature

Key result

Run / experiment:

other: 1

Parameter:

other: % Cysteine Peptide Depletion

Value:

0.892

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: % Lysine Peptide Depletion

Value:

2.306

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: Mean % Peptide Depletion (Cys + Lys)

Value:

1.599

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No or minimal reactivity

Other effects / acceptance of results:

Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

Results

 

Solvent Selection

Prior to the main test, the test substance was assessed for solubility and was found to be soluble in Isopropanol at 100mM.

 

Acceptance criteria

Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

 

Criterion		Run 1 (Cysteine)		Run 2 (Lysine)		Outcome
Std Curve r^2>0.99		0.997		0.991		PASS
PC 60.8% to 100% depletion Cys		64.698		N/A		PASS
PC 40.2% to 69.0% depletion Lys		N/A		52.256		PASS
SDCys Depletion PC<14.9%		10.909		N/A		PASS
SDLys Depletion PC<11.6%		N/A		0.991		PASS
RefA Mean Conc 0.50 ± 0.05mM		0.556*		0.533		PASS*/PASS
Peak Area CV RefB <15.0%		8.970		0.377		PASS
Peak Area CV RefC <15.0%		5.137		0.844		PASS
SDCys Depletion Test Substance <14.9%		1.546		N/A		PASS
SDLys Depletion Test Substance <11.6%		N/A		0.332		PASS
RefC Mean Conc 0.50 ± 0.05mM		0.547		0.528		PASS

Cys = Cysteine, Lys = Lysine, SD = Standard Deviation, CV = Coefficient of Variation, PC = Positive Control. *Passed as acceptable by Study Director.

 

Results Summary

The test substance produced 1.599% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. A single HPLC analysis for both the Cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. No co-elution (peak areas produced at 220nm with the same retention time as the peptide) was observed for this test substance.

 

Name	Test Substance ID	% Cysteine Peptide Depletion	% Lysine Peptide Depletion	Mean % Peptide Depletion (Cys + Lys)	DPRA Prediction	DPRA Reactivity Class
Acetamide MEA	TA4	0.892	2.306	1.599	Non-Sensitiser	No or Minimal Reactivity

 

Deviations

During the initial runs there was an HPLC system failure and therefore the study was halted (data was retained) and the study was repeated once a new system had been installed and validated.

Conclusion

The final mean % peptide depletion observed in the DPRA was 1.599%. Therefore, Acetamide MEA was classified as a non-sensitiser with no or minimal reactivity as per the Cysteine 1:10 / Lysine 1:50 prediction model.

Interpretation of results:

other: Not classified based on EU CLP Criteria

Conclusions:

Under study conditions, the test substance was determined to be non-sensitiser to the skin.

Executive summary:

A study was conducted to determine the skin sensitisating potential of the test substance usingDirect Peptide Reactivity Assay (DPRA), according to OECD Guideline 442C, in compliance with GLP. Test substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an high performance liquid chromatography (HPLC) system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. Test substance was compared to vehicle controls containing the test substance solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes. The test substance produced 1.599% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. A single HPLC analysis for both the cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance was concluded to be non-sensitiser to the skin (XCellR8, 2018).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 12, 2018 to Janaury 24, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT)

Version / remarks:

EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015)

Deviations:

no

Principles of method if other than guideline:

The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 h exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. 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 marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Details on the study design:

Description of the test method
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-h exposure to the test substance. 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.

Characterisation of the test method
The h-CLAT has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Method workflow summary
Solubility was first determined for the test substance using either culture medium (RPMI 1640) or DMSO. Note that for this method, test substances with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test substances with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid. THP-1 cells were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test substance that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs. THP-1 cells were pre-cultured for 72 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test substance. This dilution range was used to dose the cells again for 24 ±0.5 h. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Test system material source
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived.

OECD test Guideline relating to the test method
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 09 Oct 2017.

XCellR8 SOP
The study was performed using the h-CLAT method as detailed in OECD TG 442E and also in EURL ECVAM DB-ALM Protocol No. 158 (Issued 02 Mar 2017). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data is collected using XCellR8 internal protocols (IPs).

Key result

Run / experiment:

other: Average of two run

Parameter:

other: CV75 (µg/mL)

Remarks:

A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)

Value:

5 000

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Average of two representatives

Parameter:

other: CD54 expression measured as RFI (up to 5 mg/mL, top dose concentration)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Average of two representatives

Parameter:

other: CD86 expression measured as RFI (up to 5 mg/mL, top dose concentration)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

Results

 

a) Solvent Selection and CV75 Determination

Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in culture medium at 500 mg/mL. The CV75 value derived from two independent experiments was as follows:

 

CV75 (Rep1)	CV75 (Rep2)	Average CV75
>5000	>5000	>5000 µg/mL (> 5mg/mL)

 

b) Acceptance Criteria

Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. 

CV75 Determination
Criterion	Run 1	Run 2	Outcome
Cell viability must be ≥ 75% at the lowest dose.	97.67	96.71	PASS
The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.	96.70; max dose (5mg/mL) used.	95.61; max dose (5mg/mL) used.	PASS

 

Measurement of CD54 and CD86 Expression
Criterion	Run 1	Run 2	Outcome
Cell viabilities of medium and solvent controls should be higher than 90%	98.19	98.18	PASS
In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control	No solvent control	No solvent control	N/A
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%	CD54: 142.87 CD86: 143.58	CD54: 150.99 CD86: 139.91	PASS
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%.	CD54 RFI: 327 CD54 Via: 90.38 CD86 RFI: 248 CD86 Via: 90.93	CD54 RFI: 239 CD54 Via: 93.00 CD86 RFI: 167 CD86 Via: 91.41	PASS
For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run	8/8	8/8	PASS
Negative results are acceptable only for test substances exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.	97.68 (5mg/mL used as max dose)	97.34 (5mg/mL used as max dose)	PASS

RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity, DMSO - Dimethyl Sulphoxide

 

 

Results Summary

The CV75 dose informs the dosing range selected for the CD54/86 expression assay. The top dose (5mg/mL) was non-cytotoxic so this concentration was taken forward as the top concentration for the CD54/86 expression assay. The following tables show the expression of CD54 and CD86 against test substance dose with concurrent cytotoxicity measurement:

 

Run 1 (Valid): Result = Non-Sensitiser

Test Substance Dose (µg/mL)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
5000.00	97.12	97.78	98.15	97.68	18	11
4166.67	97.64	98.20	98.03	97.96	59	58
3472.23	97.64	97.29	97.20	97.37	42	37
2893.52	97.28	97.69	97.42	97.46	49	43
2411.27	97.91	97.20	97.11	97.41	145	104
2009.39	97.74	97.97	96.55	97.42	78	54
1674.49	98.40	97.76	96.74	97.63	70	29
1395.41	97.24	96.94	95.95	96.71	129	56

 

Run 2 (Valid): Result = Non-Sensitiser

Test Substance Dose (µg/mL)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
5000.00	97.46	98.13	96.44	97.34	105	108
4166.67	97.40	97.26	95.66	96.77	74	87
3472.23	96.60	95.92	94.98	95.83	82	133
2893.52	97.66	97.05	96.45	97.05	55	73
2411.27	97.27	96.16	97.04	96.82	86	108
2009.39	97.99	98.13	97.19	97.77	59	88
1674.49	97.53	97.77	97.48	97.59	84	112
1395.41	98.21	97.88	97.28	97.79	99	126

 

As can be seen from the data, the expression of CD54 as measured by the RFI did not cross the threshold (RFI ≥200) at any of the doses tested. The expression of CD86 as measured by the RFI did not cross the threshold (RFI ≥150) at any of the doses tested. As the CD54/CD86 expression did not cross the threshold in either run the test substance is classified as a Non-Sensitiser. Cell viability did not fall below 50% at any of the test substance concentrations.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be non-sensitising to skin.

Executive summary:

An in vitro study was conducted to determine the skin sensitising potential of the test substance using the Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C, in compliance with GLP. The substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in a solution at 100 mM in combination with either cysteine or lysine-containing peptides, then run on a High Performance Liquid Chromatography (HPLC) system with UV detection to measure peptide concentrations. The test substance was compared to vehicle controls containing solvent in combination with either cysteine or lysine in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns the test substances to one of four reactivity classes. The substance produced 1.599% mean cysteine and lysine peptide depletion Using the cysteine 1:10 / lysine 1:50 prediction model, the test substance was therefore classified as a non-sensitiser, with no or minimal reactivity. A single HPLC analysis for both the cysteine and the lysine peptides was considered sufficient as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of Ref A for cysteine which was marginally outside the range (0.556 mM, range 0.45 mM to 0.55 mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance was concluded to be non-sensitising to skin (XCellR8, 2018).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 12, 2018 to February 15, 2018

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Test and Reference substances

Test substance
Concentration tested: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977 µM
Lot no: 0000974911
Solvent: 1% Ethanol in cell culture medium
Purity: 100%

Positive control
Substance name: Cinnamic Aldehyde
Concentration tested: 8µM to 128 µM
Lot no: STBG0250V
Expiry date: Jul 2022
Solvent: 1% Ethanol in cell culture medium
Purity: ≥99%

Negative control
Substance name: Ethanol
Lot no: SHBJ3722 for repetition 1 and 2, SHBJ2760 for repetition 3
Concentration tested: 1%
Purity: ≥99.5%
Expiry date: 17 Nov 2022 for repetition 1 and 2, 13 Feb 2023 for repetition 3

Key result

Run / experiment:

other: All concentrations tested (3 repetitions)

Parameter:

other: EC1.5 value

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 250µM concentration

Parameter:

other: Imax

Remarks:

Maximum-fold induction observed within the concentration range tested

Value:

1.41

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 and 3. However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.

Results

Solubility Assessment

The test concentrations of test substance used in the KeratinoSens^TM test were selected on the basis of solubility test carried out prior to the study. Solubility of test substance in cell culture medium (confirmed up to 200 mM in Ethanol; subsequent dilution in cell culture medium giving a top concentration of 2000 µM).

Determination of the skin sensitisation potential of test substance 

REP 1	Test substance concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250	500	1000	2000
Mean of fold induction	0.768	0.815	0.838	0.932	1.070	1.023	1.007	1.040	0.953	1.031	1.073	0.967
SD	0.037	0.061	0.082	0.030	0.014	0.079	0.059	0.037	0.092	0.101	0.004	0.054
Viability %	136.72	117.43	112.65	109.39	107.00	104.20	109.45	106.14	110.52	102.49	105.37	115.00
Imax	1.073 at 1000µM
EC1.5	No EC1.5 was determined as induction value at 2000µM was 0.967
IC50	>2000µM as viability value at 2000µM was 115%
IC30	>2000µM as viability value at 2000µM was 115%

  

REP 2	Test substance concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250	500	1000	2000
Mean of fold induction	1.405	0.868	1.325	1.212	1.114	1.038	1.255	1.204	1.248	1.380	1.143	0.625
SD	0.171	0.156	0.049	0.084	0.206	0.134	0.064	0.100	0.049	0.144	0.161	0.038
Viability %	99.21	115.16	83.74	93.42	90.45	85.64	90.26	87.49	90.33	92.11	100.34	109.56
Imax	1.405 at 0.977µM
EC1.5	No EC1.5 was determined as induction value at 2000µM was 0.625
IC50	>2000µM as viability value at 2000µM was 109.56%
IC30	>2000µM as viability value at 2000µM was 109.56%

  

REP 3	Test substance concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250	500	1000	2000
Mean of fold induction	1.129	1.009	1.101	1.171	1.070	1.198	0.984	1.243	1.410	1.158	1.065	1.112
SD	0.084	0.136	0.139	0.196	0.159	0.147	0.242	0.105	0.200	0.139	0.164	0.192
Viability %	112.06	82.48	78.96	74.82	70.55	73.86	88.80	78.70	85.94	91.39	106.63	118.96
Imax	1.410 at 250µM
EC1.5	No EC1.5 was determined as induction value at 2000µM was 1.112
IC50	>2000µM as viability value at 2000µM was 118.96%
IC30	>2000µM as viability value at 2000µM was 118.96%

 

Determination criteria for the skin sensitisation potential of test substance
	REP1	REP2	REP3
Does at least one concentration of test substance induce 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 EC1.5 value occur at a concentration <1000 µM (or <200 µg/ml)	N/A	N/A	N/A
Does the test substance induce the luciferase in a dose-dependent manner	N/A	N/A	N/A
Classification	Non-Sensitiser	Non-Sensitiser	Non-Sensitiser

N/A: Not applicable

 

Assay Acceptance Criteria (Mean of the 3 repetitions) 

Criteria	Result	Pass or Fail
1-Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in at least one concentration	Yes	Pass
2-Average induction of PC at 32µM is [1.6-3.0]	No (1.36)	Fail*
3-EC1.5value is [6-39µM]	No (53.18)	Fail*
4-CV% of blank values < 20%	Yes (14.51)	Pass

*Acceptance Criterion 2 and 3 are not met, however, all other acceptance criteria are met and there is dose-dependent increase of induction with the Positive Control, with 2 concentrations above the induction threshold. Therefore, the results are considered as valid.

 

 

Discussion

The human skin sensitisation potential of test substance was assessed using validated in vitro method: the KeratinoSens^TM 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. The adapted method showed full concordance with the Validated Reference Method (VRM) – the KeratinoSens^TM standard protocol. We recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, is included in the dossier.

 

In this study, test substance was classified as non-sensitiser to human skin.

 

The sensitisation potential of test substance was quantified by calculating 2 parameters known as the EC_1.5 and the I_MAX value. The meanings of these are as follows:

 

1) The EC_1.5 value means the Effective Concentration (EC) of test substance that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls. If at least one concentration induces luciferase activity to >1.5, then the product is classified as a skin sensitiser. (Note: this classification also requires the cell viability measured by MTT to be greater than 70%). Test substance did not cause luciferase induction >1.5 in any of the 3 repetitions. Therefore, test substance was classified as a non-sensitiser.

 

2) The I_Max value is the maximum-fold induction observed within the concentration range tested. Although the KeratinoSens^TM test is not validated to predict potency, the I_MAX value can provide a useful tool for a very preliminary comparison of sensitisation potential between test substances. The maximum induction was observed at a test concentration of 1000µM, which showed an I_max value of 1.073 in repetition 1; 1.405 at 0.977µM in repetition 2; 1.410 at 250µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced I_max values of up to 36-fold over untreated controls.

 

All of the formal acceptance criteria of the tests were met except foracceptance criterion 2 and 3. However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was concluded to be non-sensitising to skin.

Executive summary:

An in vitro study was conducted to determine skin sensitisation potential of the test substance (purity: assumed to be >96%), using the ARE-Nrf2 Luciferase test method (KeratinoSensTM), according to OECD Guideline 442D, in compliance with GLP. After 48 h of exposure of cells to 12 test concentrations of the test substance (i.e., at 0.977, 1.953, 3.906, 7.813, 15.625, 31.25, 62.5, 250, 500, 1000 and 2000 µM), 5 concentrations of cinnamic aldehyde (8µM to 128 µM), as positive control and 1% Ethanol, as negative control, the luciferase measurements and MTT viability testing were performed. Three repetitions were performed and each repetition consisted of 3 x 96-well plates for luminescence and 2x 96-well plates for MTT. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5 (effective concentration of test substance that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls) and Imax value (maximum-fold induction observed within the concentration range tested). In each repetition of the study, no concentration of the test substance caused luciferase induction ≥1.5. Therefore, the test substance was classified as non-sensitiser. The maximum induction was observed at a test concentration of 250 µM, which showed an Imax value of 1.410. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 and 3. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered valid. Under the study conditions, the test substance was concluded to be non-sensitising to skin (XcellR8, 2018). 

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 25, 1977 to September 02, 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: generally accepted scientific principles and well documented study details, acceptable for assessment

Deviations:

no

GLP compliance:

no

Type of study:

patch test

Justification for non-LLNA method:

- Human study
- Before LLNA testing

Species:

other: Human, healthy volunteers

Sex:

not specified

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

7.5% test substance preparation

Day(s)/duration:

1 - 4 d

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

7.5% test substance preparation

Day(s)/duration:

24 h, then follow-up readings at 24, 48 and 72 h

Adequacy of challenge:

other: sensitisation responses

No. of animals per dose:

50 human volunteers

Details on study design:

In the induction phase, a 7.5% test substance preparation (in distilled water) was applied epicutaneously on 50 healthy volunteers for 24 h under an occlusive type of coverage. The applications were done daily on Monday, Tuesday, Wednesday and Thursday for 3 consecutive weeks. After a rest period of ca. 2 weeks, the test substance was applied with the same conditions on a new site as a challenge. A the end of 24 h, the occluded patch was removed and the site was read for immediate response. Follow-up readings were made 24, 48 and 72 h later. The skin effects were then scored for irriation (erythema, edema as well as other irritation signs).

Challenge controls:

-

Positive control substance(s):

no

Positive control results:

-

Key result

Reading:

other: Overall

Hours after challenge:

24

Group:

test chemical

Dose level:

7.5% test substance preparation

No. with + reactions:

0

Total no. in group:

50

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: Overall

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

not measured/tested

Key result

Reading:

other: Overall

Hours after challenge:

24

Group:

positive control

Dose level:

0

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

not measured/tested

During the second week of the induction period, visible irritation (slight erythema) was observed in one individual. During week 3, irritation was recorded in 2 individuals (slight to marked erythema). This was considered as a manifestation of skin fatigue resulting from the cummulative effect of 11 or more applications. No visible irritation was observed in any of the 50 individuals following challenge.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be non-sensitising to skin

Executive summary:

An in vivo study was conducted to determine the skin sensitisation potential of the test substance (purity not specified) according to the Human Repeated Insult Patch Test (HRIPT). In the induction phase, a 7.5% test substance preparation (in distilled water) was applied epicutaneously to 50 healthy volunteers for 24 h under an occlusive type of coverage. The applications were done daily on Monday, Tuesday, Wednesday and Thursday for 3 consecutive weeks. After a rest period of ca. 2 weeks, the substance was applied under the same conditions to a new site as a challenge. At the end of 24 h, the occluded patch was removed and the site was read for immediate response. Follow-up readings were made 24, 48 and 72 h later. The skin effects were then scored for irriation (erythema, edema as well as other irritation signs). During the second week of the induction period, visible irritation (slight erythema) was observed in one individual. During Week 3, irritation was recorded in 2 individuals (slight to marked erythema). This was considered as a manifestation of skin fatigue resulting from the cumulative effect of 11 or more applications. No visible irritation or skin sensitisation response was observed in any of the 50 individuals following challenge, confirming that the test substance was not a skin sensitiser under the study conditions (Product Investigation Inc., 1977).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Study 1:

An in vitro study was conducted to determine the skin sensitising potential of the test substance using the Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C, in compliance with GLP. The substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in a solution at 100 mM in combination with either cysteine or lysine-containing peptides, then run on a High Performance Liquid Chromatography (HPLC) system with UV detection to measure peptide concentrations. The test substance was compared to vehicle controls containing solvent in combination with either cysteine or lysine in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns the test substances to one of four reactivity classes. The substance produced 1.599% mean cysteine and lysine peptide depletion Using the cysteine 1:10 / lysine 1:50 prediction model, the test substance was therefore classified as a non-sensitiser, with no or minimal reactivity. A single HPLC analysis for both the cysteine and the lysine peptides was considered sufficient as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of Ref A for cysteine which was marginally outside the range (0.556 mM, range 0.45 mM to 0.55 mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance was concluded to be non-sensitising to skin (XCellR8, 2018).

 

Study 2:

An in vitro study was conducted to determine the skin sensitisation potential of the test substance (purity: assumed to be >96%) using the Human Cell Line Activation Test (h-CLAT) method according to OECD Giudeline 442E, in compliance with GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (i.e., 39, 78, 156, 312.5, 625, 1250, 2500 and 5000 µg/mL) and incubated for 24 ± 0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be >5000 µg/mL. For the test substance, up to highest tested concentration, CD54 and CD86 expression as Relative Fluorescence Intensity (RFI) were found to be <200 and <150, respectively. As the CD54/CD86 expression did cross the sensitisation threshold (CD54≥200 and CD86≥150), the test substance was classified as non-sensitising to skin. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Cell viability did not fall below 50% at any of the test substance concentrations. Under the study conditions, the test substance was determined to be non-sensitising to skin (XcellR8, 2018).

 

Study 3:

An in vitro study was conducted to determine skin sensitisation potential of the test substance (purity: assumed to be >96%), using the ARE-Nrf2 Luciferase test method (KeratinoSensTM), according to OECD Guideline 442D, in compliance with GLP. After 48 h of exposure of cells to 12 test concentrations of the test substance (i.e., at 0.977, 1.953, 3.906, 7.813, 15.625, 31.25, 62.5, 250, 500, 1000 and 2000 µM), 5 concentrations of cinnamic aldehyde (8µM to 128 µM), as positive control and 1% Ethanol, as negative control, the luciferase measurements and MTT viability testing were performed. Three repetitions were performed and each repetition consisted of 3 x 96-well plates for luminescence and 2x 96-well plates for MTT. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5 (effective concentration of test substance that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls) and Imax value (maximum-fold induction observed within the concentration range tested). In each repetition of the study, no concentration of the test substance caused luciferase induction ≥1.5. Therefore, the test substance was classified as non-sensitiser. The maximum induction was observed at a test concentration of 250 µM, which showed an Imax value of 1.410. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 and 3. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered valid. Under the study conditions, the test substance was concluded to be non-sensitising to skin (XcellR8, 2018). 

Study 4:

An in vivo study was conducted to determine the skin sensitisation potential of the test substance (purity not specified) according to the Human Repeated Insult Patch Test (HRIPT). In the induction phase, a 7.5% test substance preparation (in distilled water) was applied epicutaneously to 50 healthy volunteers for 24 h under an occlusive type of coverage. The applications were done daily on Monday, Tuesday, Wednesday and Thursday for 3 consecutive weeks. After a rest period of ca. 2 weeks, the substance was applied under the same conditions to a new site as a challenge. At the end of 24 h, the occluded patch was removed and the site was read for immediate response. Follow-up readings were made 24, 48 and 72 h later. The skin effects were then scored for irriation (erythema, edema as well as other irritation signs). During the second week of the induction period, visible irritation (slight erythema) was observed in one individual. During Week 3, irritation was recorded in 2 individuals (slight to marked erythema). This was considered as a manifestation of skin fatigue resulting from the cumulative effect of 11 or more applications. No visible irritation or skin sensitisation response was observed in any of the 50 individuals following challenge, confirming that the test substance was not a skin sensitiser under the study conditions (Product Investigation Inc., 1977).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in vitro studies, confirmed by an HRIPT, the test substance does warrant classification for skin sensitisation according to EU CLP criteria (Regulation 1272/2008/EC).