Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Endpoint summary

Description of key information

The test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was negative in the DPRA and the KeratinoSens assay, but positive in the h-CLAT. Overall, following the “2 out of 3” prediction model, the test item is regarded to be a non-sensitiser

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

Study period:

12 October 2018 - 15 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

see "Any other information on materials and methods incl. tables"

Positive control results:

The positive control gave positive results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50% in each run.

Parameter:

other: CV75 (µg/mL)

Value:

17.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Parameter:

other: EC200 for CD54 expression (µg/mL)

Value:

9.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Parameter:

other: EC150 for CD86 expression (µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no clear dose-response was observed for CD86 expression, the effective concentration (EC150) corresponding to CD86 expression could not be determined

Preliminary tests (Dose finding assays)

Two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control. For the first run the default 100 mg/mL stock solution was used for the test item dissolved in saline. This stock concentration corresponded to 1000 µg/mL as the highest final test item concentration on the plate.Due to low cell viability observed in the first run, for the second runlower stock concentration was used,which was 3.1 mg/mL. The highest final concentration on the plate was 30 µg/mL.

In the first run a 2-fold serial dilution was used when preparing the master solutions, but in the second run a 1.2-fold serial dilution was used in order to be able todetermine the CV75 value more accurately.

 

Table 4.a         Dose finding test results

Date	Test dose (µg/mL)	8	16	31	63	125	250	500	1000
23-24. October 2018	Viability (%)	91,5	76,4	37,1	6,9	0,4	1,7	37,6	58,7
Date	Test dose (µg/mL)	8,4	10,0	12,1	14,5	17,4	20,8	25	30
24-25. October 2018.	Viability (%)	94,0	93,1	90,9	86,7	79,5	69,0	57,6	56,2

 

Table 4.b         Dose finding test results

Test dose (µg/mL)	16	31		Log CV75	CV75
Viability (%)	76,4	37,1		1,21	16,4
Test dose (µg/mL)	17,4	20,8		Log CV75	CV75
Viability (%)	79,5	69,0		1,27	18,8
AVERAGE					17,6

 

CV75 values could be determined by log-linear interpolation based on the concentrations causing cell viabilities to lower close to 75%. The average CV75 value of the two runs (17.6 µg/mL) was used for setting the dose-range for measuring CD86 and CD54 expression in the main test.

 

Eight final concentrations (µg/mL) were used for the test item tested of the main test. These are (nominal concentrations):

 

1.2 × CV75 (21 µg/mL);

1 × CV75 (18 µg/mL);

1/1.2 × CV75 (15 µg/mL);

1/1.2² × CV75 (12 µg/mL);
1/1.2³ × CV75 (10 µg/mL);

1/1.2⁴ × CV75 (9 µg/mL); 

1/1.2⁵× CV75 (7 µg/mL);

and 1/1.2⁶× CV75 (6 µg/mL).

 

Main tests (CD86 and CD54 expression)

The CD86/54 expression was measured shortly after determining CV75, using the same batch of THP-1 cells.

For CD86/CD54 expression measurement, the test item was tested in three independent runs to derive a single prediction (positive/negative). Each independent run was performed on a different day.

 

The relative fluorescence intensity (RFI) was calculated for the test item, positive and negative controls for each concentration in each run for both surface markers, using the geometric mean fluorescence intensities.

 

Negative and positive control

The positive control gave positive results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50% in each run. 

The DMSO controls had negative outcomes compared to the medium control for both markers in all runs, meaning that the RFI values of both CD86 and CD54 expression was never over the positive criteria.

The cell viabilities of medium and DMSO controls were higher than 90% in all runs (taken cell viabilities of the IgG1 isotypic control). For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105%.

 

Table 5.           Positive and negative control data

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	sample	concentration	RFI		viability (IgG1)
			CD86	CD54	IgG
Exposure date:   31 October 2018	DMSO	0.2%	111	79	93,5
	DNCB	4.2 μg/mL	364	588	70,9
Exposure date:   06 November 2018	DMSO	0.2%	99	63	94,9
	DNCB	4.2 μg/mL	635	368	75,0
Exposure date:   15 November 2018	DMSO	0.2%	104	72	90,2
	DNCB	4.6 μg/mL	296	406	67,1

 

All runs were considered valid, since all runs have met the acceptance criteria stated above.

 

Test item

 

For the test item, the relative fluorescence intensity (RFI) of CD86 was greater than 150 % at some tested doses (with cell viability > 50 %) in the first and second run but not in the third run. No clear dose-response could be observed.

 

The RFI values for CD54 expression were greater than 200 % consequently at higher tested doses (with cell viability > 50 %) at all three independent runs and a clear dose response curve was presented with increasing concentrations and increasing RFI values.

 

Test item	Obtained CV75 value (µg/mL)	Result of the individual runs for CD86 (positive/negative)			h-CLAT prediction for CD86 expression	Result of the individual runs for CD54 (positive/negative)			h-CLAT prediction for CD54 expression
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	17.6	p	p	n	positive	p	p	p	positive

Table 6.        Outcome of the individual runs of the main tests

 

n – negative outcome of a valid run

p- positive outcome of valid run

 

Since 3 out of 3 runs were positive for CD54 marker expression, and 2 out of 3 runs were positive and for CD86 marker expression, the overall outcome of the study was concluded as positive.

The test itemgave positive results for CD86 and CD54 too.Since no clear dose-response was presented for CD86 expression, the effective concentration (EC150) corresponding to CD86 expression could not be determined. But aclear dose-response was presented for CD54 expression, therefore the effective concentration value (EC200) was calculated(9.8 µg/mL, 8.1 µg/mL and 10.5 µg/mL in the first, second and third runs respectively).

 

Table 7.            Effective concentrations for the test item

 

Test item	EC150 (µg/mL)	EC200 (µg/mL)
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	-	9.8

 

 

Interpretation of results:

other: expert judgement

Remarks:

positive in this assay; however, to conclude in the endpoint skin sensitisation, further studies covering MIE and key event 2 of the Adverse Outcome Pathway are taken into account.

Conclusions:

Based on the results and the h-CLAT prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was concluded positive and demonstrated a sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

In the course of this study the skin sensitization potential of “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was studied.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The averagetest item concentration that results in 75% cell viability compared to the solvent/vehicle control was 17.6 µg/mL. This value was used for setting the dose‑range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 23 µg/mL and 6 µg/mL.

The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with >50 % of cell viability) compared to the respective negative controls in two out of three runs. However the results were positive for CD86 expression, no clear dose-response could be observed, therefore the effective concentration (EC150) corresponding to CD86 expression could not be determined.

Also, the increase in CD54 marker expression (RFI) was greater than 200 % consequently at higher concentrations (with >50 % of cell viability) compared to the respective negative controls in all three independent run. A clear dose-response was presented for CD54 expression, therefore the effective concentration (EC200) was determined. The mean EC200 value for CD54 was 9.8 µg/mL.

Since the CD54 marker gave positive result at higher concentrations in all three independent runs and CD86 marker gave positive result at some tested dose in 2 out of 3 runs, the overall h-CLAT prediction was concluded positive, as well.

 

Table 1.           Summary of the h-CLAT results for the test item

Name of the Test item	Obtained CV75 value (µg/mL)	h-CLAT result for CD86 (positive/ negative)	h-CLAT result for CD54 (positive/ negative)	Obtained EC200 value (µg/mL)	h-CLAT result obtained (sensitizer/ non-sensitizer)
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	17.6	positive	positive	9.8	sensitizer

 

Based on these results and the h-CLAT prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was concluded positive and demonstrated a sensitizing potential under the experimental conditions of human Cell Line Activation Test.

However, to conclude in the endpoint skin sensitisation, further studies covering MIE and key event 2 of the Adverse Outcome Pathway are taken into account. 

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2018 - 11 October 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))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Formulation of the Test Item
The test item was formulated and examined in the test as follows: first the solubility of the test item was evaluated at the concentration of 100 mM. Acetonitrile did not dissolve the test item at all. Therefore, the test item was tried to be dissolved in ultrapure water and a homogenous, clear solution was formed.
Moreover, the compatibility of the formulation was assessed with the buffers used in the experiment (phosphate buffer and acetate buffer – both prepared as described in 5.5.1) and no precipitation was observed with either of the buffers.
Since ultrapure water is the first preferred vehicle after acetonitrile according to the guideline [3] and the formulation complied with all obligations of the guideline, it was chosen as the appropriate vehicle for the study and solubility in other vehicles was not evaluated.

Positive Control : CINNAMALDEHYDE
Synthetic Peptides:
Cysteine peptide
Name:RFA7-C
Lot:11180.02
Storage:at -20°C or below
Purity:95.03 %
Molecular weight:750.87 g/mol
Sequence:Ac-RFAACAA

Lysine peptide
Name:RFA7-K
Lot:11181.01
Storage:at -20°C or below
Purity:96.75 %
Molecular weight:775.90 g/mol
Sequence:Ac-RFAAKAA

HPLC System Conditions
HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Column temperature: 30°C
Sample temperature: 25°C
Detector: 220 nm (258 nm)
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient , 0.35 mL / min

Time A phase (%) B phase (%)
0 min 90 10
10 min 75 25
11 min 10 90
13 min 10 90
13.5 min 90 10
20 min gradient ends


Demonstration of Proficiency
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

Parameter:

other: Obtained mean % cysteine peptide depletion

Value:

0.89

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Obtained mean % lysine peptide depletion

Value:

0.18

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Mean % obtained peptide depletion

Value:

0.54

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.49 mM for the cysteine peptide and 0.51 mM for the lysine peptide. Data for the reference control A sample replicates are presented in Table 6 and 7. 

A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions standards from the peptide stock solutions. Calibration standard points were analysed by linear regression. Means of the peak areas versus the concentrations of both peptides showed good linearity with r²= 0.999 for the cysteine peptide and r²= 1.000 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM.  

All validity criteria were within acceptable limits and therefore the study can be considered valid. 

 

Table 6. Reference control A replicates for cysteine peptide 

	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV   %	Mean (mM)	SD	CV %
ref A I	2138615	0.48	2195595	4	0.49	0.0187	4
ref A II	2290737	0.52
ref A III	2157432	0.49

 

Table 7. Reference control A replicates for lysine peptide 

	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV   %	Mean (mM)	SD	CV %
ref A I	2387592	0.51	2373758	1%	0.51	0.0026	1%
ref A II	2363978	0.50
ref A III	2369703	0.50

Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion. The mean cysteine peptide concentration of the reference control C replicates was 0.48 mM and the mean lysine peptide concentration of the reference control C replicates were 0.49 mM, which were within the acceptable 0.50 ± 0.05 mM range.  

 

Moreover, the CV % for the nine reference control B and C replicates in acetonitrile and water respectively were much smaller than the acceptable 15 % for both peptides, since it was 2 % for cysteine and 1 % for lysine peptides. All validity criteria were within acceptable limits and therefore the study can be considered valid. 

 

Table 8. Reference control B and C replicates for cysteine peptide 

Name, replicate number	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV   %	Mean (mM)	SD	CV %
ref B I	2266292	0.51	2183241	2	0.49	0.0120	2
ref B II	2219742	0.50
ref B III	2239767	0.50
ref B I / 2	2179020	0.49
ref B II / 2	2144613	0.48
ref B III / 2	2154257	0.48
ref C I upw	2203592	0.50
ref C II upw	2138614	0.48
ref C III upw	2103271	0.47

 

Table 9. Reference control B and C replicates for lysine peptide 

Name, replicate number	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV   %	Mean (mM)	SD	CV %
ref B I	2375890	0.51	2349237	1%	0.50	0.0073	1%
ref B II	2366746	0.50
ref B III	2398674	0.51
ref B I / 2	2356111	0.50
ref B II / 2	2334886	0.50
ref B III / 2	2374121	0.51
ref C I upw	2334511	0.50
ref C II upw	2309891	0.49
ref C III upw	2292301	0.49

 

 

Cysteine and lysine depletion and mean peptide depletion of the test item 

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 75.36 % and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 1.95 % and 0.84 % for the cysteine and lysine depletion, respectively. Thus, this validity criterion was also met.  

The percent cysteine peptide depletion with the test item was 0.89 % while the percent lysine peptide depletion with the test item was 0.18 %. The standard deviation for the test chemical replicates was 3.25 % for the percent cysteine depletion and 2.86 % for the percent lysine peptide depletion. In Appendix II for the test chemical, the peptide peak areas of each replicate, their mean and CV %, the peptide depletion values for each replicate, their mean and SD and description of all relevant observations (solubility, precipitate, co-elution) are shown. 

 

Table 10. Cysteine peptide depletion values for the positive control and the test item  

Name, replicate number	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide depletion
			%	SD  (%)
ref C, rep I upw	2203592	0.50	-	-
ref C, rep II upw	2138614	0.48
ref C, rep III upw	2103271	0.47
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep I	2144625	0.48	2.68	3.25
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep II	2214796	0.50	-3.56*
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep III	2146421	0.48	-2.05*
CINNAMALDEHYDE, rep I	544707	0.12	75.28	1.95
CINNAMALDEHYDE, rep II	484508	0.11	77.34
CINNAMALDEHYDE, rep III	558365	0.13	73.45

 

*Negative depletion values are substituted with zero when calculating mean peptide depletion 

 

Table 11. Lysine peptide depletion values for the positive control and the test item  

Name, replicate number	Peptide   peak area   at 220 nm	Peptide conc. calculated (mM)	Peptide depletion
			%	SD  (%)
ref C, rep I upw	2334511	0.50	-	-
ref C, rep II upw	2309891	0.49	-
ref C, rep III upw	2292301	0.49	-
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep I	2334524	0.50	0.00	2.86
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep II	2417655	0.51	-4.67*
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, rep III	2280136	0.49	0.53
CINNAMALDEHYDE, rep I	1011241	0.22	56.68	0.84
CINNAMALDEHYDE, rep II	965360	0.21	58.21
CINNAMALDEHYDE, rep III	989280	0.21	56.84

 

*Negative depletion values are substituted with zero when calculating mean peptide depletion 

Table 12. Mean peptide depletion values for the positive control and the test chemical 

Name, replicate number	Obtained mean % cysteine peptide depletion	Obtained mean % lysine peptide depletion	Mean % obtained peptide depletion
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	0.89	0.18	0.54
CINNAMALDEHYDE	73.36	57.24	65.3

 

Assigning the test chemical to a reactivity class and category 

The average percent peptide depletion was calculated for the test item. By using the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. On the basis of the prediction model, chemicals assigned to the minimal reactivity class should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity class should be classified as sensitisers. Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized had the average percent peptide depletion value of 0.54 %, thus the test item is classified as a non-sensitiser. 

Interpretation of results:

other: expert judgement

Remarks:

negative in this assay; however, to conclude in the endpoint skin sensitisation, further studies covering key events 2 and 3 of the Adverse Outcome Pathway are taken into account.

Conclusions:

The mean percent peptide depletion value of the test item was 0.54 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

Executive summary:

This study was undertaken to evaluate the skin sensitization potential of the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides.

At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent.

One individual test was conducted with both peptides, since all runs met the acceptance criteria and were considered valid. All in all, the results of the two valid runs were used for the classification of the test item.

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 75.36 % and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 1.95 % and 0.84 % for the cysteine and lysine depletion, respectively. Thus, this validity criterion was also met.

The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well.

All other validity criteria were also met, thus confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 0.89 % while the percent lysine peptide depletion was 0.18 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 0.54 %, which did not exceed the 6.38 % threshold of the applicable prediction model.

The mean percent peptide depletion value of the test item was 0.54 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

However, to conclude in the endpoint skin sensitisation, further studies covering key events 2 and 3 of the Adverse Outcome Pathway are taken into account.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2018 - 18 September 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)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

see "Any other information on materials and methods incl. tables"

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests.
In addition, the average induction in the parallel plates for Trans Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold. Although these values are out of the 2 – 8 fold induction domain, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid.
There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations, thus no IC30 or IC50 values were determined.

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

fold induction did not exceed the threshold of 1.5-fold at any concentrations; thus, no EC1.5 could be calculated

Other effects / acceptance of results:

There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at several concentrations in both tests. In the first run the IC30 and IC50 values were 4 µM and 17 µM while in the second run 9 µM and 19 µM respectively. However, the fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent runs. Therefore, both runs were concluded negative for luciferase gene induction.

For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both runs met the acceptance criteria. Each individual test contained four parallel plates of which three replicates were used for the luciferase activity induction measurements and one of them was needed formeasuring the extent of cytotoxicity induced by the test chemical.

 

Test item

There was no statistically significant induction above 1.5-fold observed at any of the concentrations in either of the runs therefore no EC1.5 values were determined for the test item. The maximal fold induction was 1.40 for the first test and 1.15 for the second test. Based on the prediction model and the above described results, both tests were concluded negative.

There was cytotoxicity (cell viability lower than 70 %) induced by the test item at most of the concentrations in the first run, therefore the concentration range was lowered in the second test. In the first run the default concentration range between 2000 µM and 1 µM was used while in the second run a lower concentration range was chosen between 500 µM and 0.2 µM due to cytotoxic effects of the test item. 

In the first run the IC30 and IC50 values were 4 µM and 17 µM while in the second run 9 µM and 19 µM respectively.

 

First test:

 

		Test item
Concentration (µM)		1	2	4	8	16	31	63	125	250	500	1000	2000
Plate ID	20180918-1117-2	1.29	1.12	0.83	0.95	1.03	1.42	0.00	-0.01	-0.01	-0.01	-0.01	-0.01
	20180918-1117-3	1.02	0.83	0.91	0.83	0.85	1.31	0.00	0.00	0.00	0.00	0.00	0.00
	20180918-1117-4	0.98	1.06	0.87	0.89	1.06	1.46	0.00	0.00	0.00	0.00	0.00	0.00
average induction		1.10	1.00	0.87	0.89	0.98	1.40	0.00	0.00	0.00	0.00	0.00	-0.01
significance		0.237	0.991	0.169	0.127	0.843	0.015	0.000	0.000	0.000	0.000	0.000	0.000
viability		96%	74%	69%	54%	53%	12%	0%	1%	3%	3%	3%	1%

 

Second test:

 

		Test item
Concentration (µM)		0.2	0.5	1	2	4	8	16	31	63	125	250	500
Plate ID	20181001-1108-2	0.97	0.98	1.42	0.74	0.51	0.83	0.66	1.22	0.00	0.00	0.00	0.00
	20181001-1108-3	0.79	0.79	0.84	0.82	0.99	0.81	0.74	1.22	0.00	0.00	0.00	0.00
	20181001-1108-4	1.20	1.16	0.89	0.89	0.83	1.21	0.90	1.02	-0.01	-0.01	-0.01	-0.01
average induction		0.99	0.98	1.05	0.82	0.78	0.95	0.77	1.15	0.00	-0.01	-0.01	-0.01
significance		0.757	0.703	0.817	0.049	0.271	0.518	0.027	0.305	0.000	0.000	0.000	0.000
viability		97%	97%	96%	85%	83%	72%	58%	13%	2%	3%	3%	4%

 

 

Negative and positive control

The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was no greater than 20 % in either of the tests (14 % and 20 % respectively).

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests.

In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold. Although these values are out of the 2 – 8 fold induction domain, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid. Average fold induction values are presented in Table 3 and 4.

There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations, thus no IC30 or IC50 values were determined.

 

Interpretation of results:

other: expert judgement

Remarks:

negative in this assay; however, to conclude in the endpoint skin sensitisation, further studies covering MIE and key event 3 of the Adverse Outcome Pathway are taken into account.

Conclusions:

Based on the results and the KeratinoSens™ prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was negative therefore having a non-sensitizing potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitization potential of the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized ” was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both runs met the acceptance criteria.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests. In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity induced by the positive control at any of the concentrations.

For the test item twelve doses were used in two independent runs. In the first run the default concentration range between 2000 µM and 1 µM was used while in the second run a lower concentration range was chosen between 500 µM and 0.2 µM due to cytotoxic effects of the test item. 

There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at several concentrations in both tests. In the first run the IC30 and IC50 values were 4 µM and 17 µM while in the second run 9 µM and 19 µM respectively. However, the fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent runs. Therefore, both runs were concluded negative for luciferase gene induction.

 

Table 1.           Summary of the KeratinoSens™ results for the test item

Name of the Test item	Significant induction above 1.5-fold (yes/no)	Average IC30 (µM)	Average IC50 (µM)	KeratinoSens™ result obtained (sensitizer/ non-sensitizer)
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized	no	6.5	18	non-sensitizer

 

Based on these results and the KeratinoSens™ prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was negative therefore having a non-sensitizing potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

However, to conclude in the endpoint skin sensitisation, further studies covering MIE and key event 3 of the Adverse Outcome Pathway are taken into account.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the assessment of the sensitising potential of the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized, a battery of in chemico and in vitro assays is available, including DPRA, KeratinoSens, and h-CLAT.

Molecular initiating event: DPRA

This study was undertaken to evaluate the skin sensitization potential of the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides. 

At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent.  

One individual test was conducted with both peptides, since all runs met the acceptance criteria and were considered valid. All in all, the results of the two valid runs were used for the classification of the test item. 

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 75.36% and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 1.95% and 0.84 % for the cysteine and lysine depletion, respectively. Thus, this validity criterion was also met.  

The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well.  

All other validity criteria were also met, thus confirming the validity of the assay. 

The percent cysteine peptide depletion value of the test item was 0.89 % while the percent lysine peptide depletion was 0.18 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10/ Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 0.54%, which did not exceed the 6.38 % threshold of the applicable prediction model.  

The mean percent peptide depletion value of the test item was 0.54 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer. 

 

Key event 2: Keratinosens

In the course of this study the skin sensitization potential of the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized ” was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both runs met the acceptance criteria.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests. In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity induced by the positive control at any of the concentrations.

For the test item twelve doses were used in two independent runs. In the first run the default concentration range between 2000 µM and 1 µM was used while in the second run a lower concentration range was chosen between 500 µM and 0.2 µM due to cytotoxic effects of the test item. 

There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at several concentrations in both tests. In the first run the IC30 and IC50 values were 4 µM and 17 µM while in the second run 9 µM and 19 µM respectively. However, the fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent runs. Therefore, both runs were concluded negative for luciferase gene induction.

 

Based on these results and the KeratinoSens™ prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was negative therefore having a non-sensitizing potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

 

 

Key event 3: h-CLAT

In the course of this study the skin sensitization potential of “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was studied.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The average test item concentration that results in 75% cell viability compared to the solvent/vehicle control was 17.6 µg/mL. This value was used for setting the dose‑range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 23 µg/mL – 6 µg/mL.

The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with >50 % of cell viability) compared to the respective negative controls in two out of three runs. However the results were positive for CD86 expression, no clear dose-response could be observed, therefore the effective concentration (EC150) corresponding to CD86 expression could not be determined.

Also, the increase in CD54 marker expression (RFI) was greater than 200 % consequently at higher concentrations (with >50 % of cell viability) compared to the respective negative controls in all three independent runs. A clear dose-response was presented for CD54 expression, therefore the effective concentration (EC200) was determined. The mean EC200 value for CD54 was 9.8 µg/mL.

Since the CD54 marker gave positive result at higher concentrations in all three independent runs and CD86 marker gave positive result at some tested dose in 2 out of 3 runs, the overall h-CLAT prediction was concluded positive, as well.

 

Based on these results and the h-CLAT prediction model, the test item “Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized” was concluded positive and demonstrated a sensitizing potential under the experimental conditions of human Cell Line Activation Test.

 

Overall conclusion

Since no single in chemico or in vitro assay is sufficient to fully address the endpoint skin sensitisation, a battery of test was applied. Although a number of methods is validated for single Key events, currently no IATA (Integrated Approach toTesting and Assessment) is formally validated. One of the most commonly used assessment approaches is the “2 out of 3” prediction model, according to which any two congruent results of the three tests summarised above determine the overall assessment.

Urbisch et al (2015) examined the specificity and sensitivity of the common “2 out of 3” prediction model and found 90% accuracy when compared to human data, which is even higher than the accuracy of the LLNA (82%). The concluded, that “in many cases positives or negatives in single assays are actually FP [false posotives] or FN [false negatives], respectively, what underlines the importance of making a majority voting”.

 

The test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was negative in the DPRA and the KeratinoSens assay, but positive in the h-CLAT.

 

The Guideline states that“membrane disruptingsubstances can lead to false positive results due to a non-specific increase of CD86 expression” (OECD TG 442E).Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized is a surface active substance (41.46 mN/m at 90% saturation concentration).

 

In addition, irritating substances – the test item is classified as Skin Irritation Cat. 2, Eye Irritation Cat. 1 – may also lead to surface marker expression and, thus, to a false positive result in the h-CLAT (Urbisch et al., 2015).

 

Overall, following the “2 out of 3” prediction model, the test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized is regarded to be a non-sensitiser

 

 

Reference

Urbisch et al., 2015. Assessing skin sensitization hazard in mice and men using non-animal

test methods. Regulatory Toxicology and Pharmacology 71 (2015) 337–351

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available