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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

291-707-5

EC Name:

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

Cas Number:

90459-62-4

Molecular formula:

C24H55N3O6S

IUPAC Name:

bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate

Test material form:

solid

Remarks:

paste

In vitro test system

Details on the study design:

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

Results and discussion

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.

In vitro / in chemico

Results

Results 1

Parameter:

other: CV75 (µg/mL)

Value:

17.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

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

Results 3

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

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.