Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Endpoint summary

Description of key information

h-CLAT (OECD 442E): The test item induces dendritic cell activation.

LuSens (OECD 442D): The test item does not activate keratinocytes.

DPRA (OECD 442C): Test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

In conclusion, the test item is not considered to be skin sensitising.

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:

weight of evidence

Study period:

2018-07-03 to 2018-07-07

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

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol 154: Direct Peptide Reactivity assay (DPRA) for skin sensitisation testing

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO Guideline 3030/99

Version / remarks:

rev.4, July 11, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The reactivity of a test chemical and synthetic cysteine or lysine containing peptides was evaluated by combining the test chemical with a solution of the peptide (reaction samples) and monitoring the remaining concentration of the peptide following 24 ± 2 hours of interaction time at room temperature (25 ± 2.5°C). The peptide is a custom material containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre. Relative concentrations of the peptide following the 24 hour reaction time were determined by HPLC with gradient elution and UV detection at 220 nm. Samples were prepared and analysed in triplicates in batches to keep the total HPLC analysis time less than 30 hours.

Positive control results:

The positive control replicates (Cinnamaldehyde) showed the expected percent peptide depletion values within acceptable limits (68.53% with cysteine peptide and 51.63 % mean percent lysine peptide depletion).

Key result

Parameter:

other: average peptide depletion of both peptides [%]

Value:

2.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL 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.

ACCEPTANCE OF RESULTS: All acceptance citeria are fulfilled.
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes (cystein peptide depletion value of 68.53% (SD 2.03%) and a mean lysine peptide depletion of 51.63% (SD 2.35%))
- Acceptance criteria met for variability between replicate measurements: Yes (SD for the test chemical replicates was 7.14% for percent cysteine depletion and 0.85% for the percent lysine peptide depletion)

Interpretation of results:

GHS criteria not met

Conclusions:

Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

Executive summary:

The skin sensitisation of the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was evaluated according to OECD guideline 442C (Direct Peptide Reactivity Assay, DPRA).

At the beginning of the assay the solubility of the test chemical was assessed and acetonitrile was chosen as the appropriate solvent with which the test item formed a homogenous solution. Concentrations of the synthetic cysteine and lysine peptides following the 24 hours incubation time were determined by HPLC with gradient elution and UV detection at 220 nm.

Cysteine and lysine depletion values were to be used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptide, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used. The threshold of 6.38 % mean percent peptide depletion supported the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA).

The positive control replicates showed the expected percent peptide depletion values within acceptable limits (68.53% with cysteine peptide and 51.63 % mean percent lysine peptide depletion). The back-calculated values of the reference control replicates were within the expected molarity concentration range of 0.50 ± 0.05 mM. The experiment was considered to be valid.

The percent cysteine peptide depletion value of Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was 3.58 % while the percent lysine peptide depletion was 1.79 %. The Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers based on the threshold of 6.38 %. The average percent peptide depletion value of the test item was 2.69 % which does not exceed the threshold. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-09-17 to 2018-10-04

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

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Cell or Test System:
The KeratinoSens™ cell line is a transgenic cell line with a stable Luciferase construct insertion.
Name: KeratinoSens™ cell line
Description: immortalized adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids
Supplier: Givaudan Schweiz AG
Lot Number: 20160415
Date of production: April 15, 2016 [7]
Storage: Vapor phase of liquid nitrogen
Maintance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 μg/mL G418.
Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418
Exposure medium: DMEM containing 1 (v/v) % FBS without G418

Positive control:
The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 μM.

Negative control:
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 7.3.1, so that the final negative (solvent) control concentration was 1% DMSO in exposure medium on the treated plates.
This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO found in the tested chemical and in the positive control.

Preparation of cells:
For testing cells were 80 - 90% confluent and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium, and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1°C in the presence of 5 % CO2.

Exposure:
After the 24 hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test chemical and control substances were added to each well in a way that an additional 4 fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4× master solution to 150 μL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1°C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test chemical.

Luciferase activity measurements:
After the 48 hour exposure time with the test chemical and control substances, cells were washed with DPBS (270 μL), and 1× lysis buffer (20 μL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

Cytotoxicity:
For the cell viability assay, medium was replaced after the 48 hour exposure time with MTT working solution (200 μL) and cells were incubated for 4 hours at 37 ± 1°C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilized by the addition of isopropanol (50 μL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

Acceptance Criteria:
For each test chemical and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements, were needed. In case of discordant results between the two independent tests, a third test containing three replicates should have been performed. Each independent test was to be performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may have come from the same passage however. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of Trans-Cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO cannot be greater than 20 % in each test which consists of 6 wells tested in triplicate.

Data evaluation:
The following parameters (endpoint values) are calculated in the KeratinoSens™ test method:
- the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
- the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained;
- the IC50 and IC30 concentration values for 50 % and 30 % reduction of cellular viability.

Prediction model:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 tests, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is equal or higher than 1.5 fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is higher than 70 % at the lowest concentration with induction of luciferase activity ≥ 1.5 fold;
- the EC1.5 value is less than 1000 μM (or < 200 μg/mL for test chemicals with no defined molecular weight);
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).

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.

Key result

Run / experiment:

other: 1st test

Parameter:

other: maximum fold Luciferase induction

Value:

1.455

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2nd test

Parameter:

other: maximum fold Luciferase induction

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, our laboratory demonstrated technical proficiency (Study Number: 392-442-4012), using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

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.

Table 1: Average fold induction, significance and viability (%) values for the positive control in the first test

Concentration (µM)	Trans-Cinnamaldehyde
	4	8	16	32	64
average induction	1.05	1.29	1.49	2.49	9.40
significance	0.636	0.004	0.066	0.002	0.000
viability	113%	125%	128%	121%	113%

 

Table 2: Average fold induction, significance and viability (%) values for the positive control in the second test 

Concentration (µM)	Trans-Cinnamaldehyde
	4	8	16	32	64
average induction	1.18	1.13	1.73	2.70	11.42
significance	0.044	0.236	0.001	0.000	0.014
viability	107%	112%	118%	123%	72%

 

Table 3: Average fold induction, significance and viability (%) values for the test item in the individual tests

Fist test 18 September - 21 September 2018

Concentration (µM)		Test item
		1	2	4	8	16	31	63	125	250	500	1000	2000
Plate ID	20180918-1117-2	0.97	1.12	1.23	1.72	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	20180918-1117-3	0.90	0.93	0.78	1.61	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	20180918-1117-4	0.93	1.21	0.95	1.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
average induction		0.93	1.09	0.99	1.45	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
significance		0.289	0.496	0.843	0.074	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
viability		98%	102%	102%	96%	0%	1%	-1%	1%	0%	0%	1%	0%

Second test 01 October - 04 October 2018

Concentration (µM)		Test item
		4	5	6	7	9	11	13	15	19	22	27	32
Plate ID	20181001-1108-2	1.01	0.99	1.02	1.34	1.38	1.15	0.03	0.00	0.00	0.00	0.00	0.00
	20181001-1108-3	0.79	0.96	1.09	0.96	1.12	1.02	0.01	0.00	0.00	0.00	0.00	0.00
	20181001-1108-4	0.82	1.03	1.16	0.95	0.95	1.10	0.65	0.00	-0.01	-0.01	-0.01	-0.01
average induction		0.87	0.99	1.09	1.08	1.15	1.09	0.23	0.00	0.00	0.00	0.00	0.00
significance		0.277	0.864	0.284	0.610	0.400	0.290	0.015	0.000	0.000	0.000	0.000	0.000
viability		103%	102%	103%	95%	95%	39%	1%	2%	1%	2%	1%	2%

Interpretation of results:

other: no activation of keratinocytes

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative therefore having a non-sensitizing potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

The skin sensitization potential of the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD guideline 442D.

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 (viability < 70 %) 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 with 1.2 fold dilutions between 32 μM and 4 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 10 μM and 12 μM while in the second run 10 μM and 11 μM were determined. 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-06-18 to 2018-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°158: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Principle of the h-CLAT Method
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 hours exposure to the test item. 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 activation process through which DCs change from antigen processing to antigen presenting cells addresses the third key event of the skin sensitization Adverse Outcome Pathway.
The changes of surface phenotypic biomarker expression were measured and quantified by flow cytometry following cell staining with fluorochrome-tagged antibodies. The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurred at sub-cytotoxic concentrations.

Preliminary tests (Dose finding assays)
Four independent dose finding assay runs were performed for the test item to determine the test item concentration that results in 75% cell viability (CV75) compared to the solvent/vehicle control. The mean of the two CV75 values was used for setting the dose-range for measuring CD86 and CD54 expression in the main test.

Main Test (CD86 and CD54 expression)
For CD86/CD54 expression measurement, the test item was tested in three independent runs to derive a single prediction (positive/negative).

Test item dilutions
DMSO was used to dissolve the test item for the stock solution (SS) in the main tests. The test item was first diluted to the concentration corresponding to 500-fold of the 1.2 × CV75 determined in the dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using DMSO (eight concentrations). The master solutions were then further diluted 250-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.

Solvent/vehicle controls
The solvent/vehicle control was prepared as in the dose finding test.

Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution.

Application of test item and control substances
For test item and control substance, at least two independent runs for CD86/CD54 expression measurement were needed to obtain a prediction. Each independent run was performed on a different day. Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 10^6 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours as described in 6.5.4.

FITC staining
After 24 ± 0.5 hours of exposure, cells were transferred from 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation. The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes.
After centrifugation, cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies at 4°C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158 were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

Flow cytometry measurement and RFI determination
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for positive control (ctrl) cells and chemical-treated cells were calculated according to the following equation:

RFI = (MFI of chemical-treated cells - MFI of chemical-treated control cells) / (MFI of solvent/vehicle ctrl cells - MFI of solvent/ vehicle-treated isotype ctrl cells) x 100

The calculated cell viabilities from the isotype control (ctrl) cells (which are stained with mouse IgG1 isotype antibodies) were also noted.
The PI uptake was analysed on channel FL-3. Cell viability was determined by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

Acceptance Criteria of the h-CLAT Method:
Requirements for qualified testing
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 and CD54 ≥ 200) and cell viability should be more than 50%.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 and CD54 > 200).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.

Abnormal values
- RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction.
- If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.

Requirement for data acceptance
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90%.
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.

Key result

Run / experiment:

other: 1 to 3

Parameter:

other: % increase in CD54 marker expression compared to negative control

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1 to 3

Parameter:

other: % increase in CD86 marker expression compared to negative control

Value:

150

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:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in OECD 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference range (Study Number: 392-442-3821). Moreover, a historical database of reactivity check results and with the positive and solvent/vehicle controls was generated and has been maintained to confirm the reproducibility of the test method over time.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Stock concentrations for CV75 determination (stock solution – SS)

A trial forming was done for the test item in DMSO, since the highest allowed 500 mg/mL stock concentration was too saturated to form a clear solution. Thus a less concentrated solution was tried by going down two-fold. At 250 mg/mL a clear, yellowish, homogenous solution was formed. The exact concentration of the test item stock solution was 250 000 μg/mL in the first CV75 determination but in the second and third CV75 determination lower stock concentration was used due to low cell viability at the higher final concentrations of the plate. The used stock concentration in the second and third run were 2500 μg/mL and 2600 μg/mL. Since the cell viability was too high, we had to perform a fourth CV75 determination where the used stock concentration was 8100 μg/mL. In the first two runs a 2 fold serial dilution was used when preparing the master solutions, while for the last two runs a narrower 1.2 fold serial dilution was used.

CD86 and CD54 expression

The relative fluorescence intensity (RFI) was calculated for the test item for each concentration in each run for both surface markers, using the geometric mean fluorescence intensities. All runs were considered valid, since all runs have met the acceptance criteria stated above. The outcome of the executed individual runs for the test item is stated in Table 1.

For CD86 expression the test item was consequently negative (RFI < 150), while regarding CD54 expression, the test item gave concordant positive results (RFI ≥ 200).

Table 1: Outcome of the individual runs of the main tests

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
Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides	13.9	n	n	n	negative	p	p	p	positive

n – negative outcome of a valid run

p – positive outcome of a valid run

EC150 and EC 200 values for sensitisers

Since the test item gave positive result for CD54, its effective concentration value (EC200) was calculated.

Table: 2. Effective concentration for the test item

Test item	EC150 (µg/mL)	E200 (µg/mL)
Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides	-	12.0

Table 3: 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) and obtained EC200 value (μg/mL)	h-CLAT result obtained (sensitizer/ non-sensitizer)
Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides	13.9	negative	positive (12.0 μg/mL)	sensitizer

 

Since the increase in CD54 marker expression (RFI) is higher than 200 % compared to the respective negative controls in at least one tested dose (>50 % of cell viability) in each independent run, the test item prediction is considered as positive. Based on these results and the prediction model, the test item demonstrated an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test, during this study.

 

Interpretation of results:

other: The test item induces dendritic cell activation.

Conclusions:

Based on these results and the h-CLAT prediction model, the test item demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

In an OECD 442E study the potential of the test item to induce dendritic cell activation was tested according to the in vitro human Cell Line Activation Test (h-CLAT). The extent of cytotoxicity induced on THP-1 cells by the test item was studied in four dose finding tests. The test item concentrations where cell viability was at 75 % (CV75 values) compared to the solvent/vehicle control were calculated by log-linear interpolation from the results of dose finding tests. The average CV75 value was 13.9 μg/mL. This concentration was used for setting the dose-range for measuring CD86 and CD54 expression in the main test (eight doses were used in each independent run between 17.0 μg/mL – 4.6 μg/mL).

Measurement of CD86 and CD54 expression was performed in three independent runs. No increase of CD86 expression was observed when the dose-levels of Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides (with cell viability > 50%) were compared with the negative control. However increase of CD54 expression at higher concentrations (with cell viability > 50%) compared with the negative control was noticed consequently in all three independent run. A positive outcome and clear dose-response were presented for CD54 expression, therefore the effective concentration (EC200) was determined by linear interpolation. The EC200 value for CD54 was 12.0 μg/mL. Since the increase in CD54 marker expression (RFI) is higher than 200 % compared to the respective negative controls in at least one tested dose (>50 % of cell viability) in each independent run, the test item prediction is considered as positive. Based on these results and the prediction model, the test item demonstrated an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test during this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. There are three in vitro/in chemico studies available with the test item assessing its skin sensitising potential. The studies were conducted according to GLP and the respective OECD TG (OECD 442C, OECD 442D, OECD 442E) and are considered valid. The available data is considered suitable and reliable to allow assessment of the test items properties in regards to skin sensitisation.

h-CLAT (OECD 442E)

In an OECD 442E study the potential of the test item to induce dendritic cell activation was tested according to the in vitro human Cell Line Activation Test (h-CLAT). The extent of cytotoxicity induced on THP-1 cells by the test item was studied in four dose finding tests. The test item concentrations where cell viability was at 75 % (CV75 values) compared to the solvent/vehicle control were calculated by log-linear interpolation from the results of dose finding tests. The average CV75 value was 13.9 μg/mL. This concentration was used for setting the dose-range for measuring CD86 and CD54 expression in the main test (eight doses were used in each independent run between 17.0 μg/mL – 4.6 μg/mL).

Measurement of CD86 and CD54 expression was performed in three independent runs. No increase of CD86 expression was observed when the dose-levels of Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides (with cell viability > 50%) were compared with the negative control. However increase of CD54 expression at higher concentrations (with cell viability > 50%) compared with the negative control was noticed consequently in all three independent runs. A positive outcome and clear dose-response were presented for CD54 expression, therefore the effective concentration (EC200) was determined by linear interpolation. The EC200 value for CD54 was 12.0 μg/mL. Since the increase in CD54 marker expression (RFI) is higher than 200 % compared to the respective negative controls in at least one tested dose (>50 % of cell viability) in each independent run, the test item prediction is considered as positive. Based on these results and the prediction model, the test item demonstrated an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test during this study.

LuSens (OECD 442D)

The skin sensitization potential of the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD guideline 442D.

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 (viability < 70 %) 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 with 1.2 fold dilutions between 32 μM and 4 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 10 μM and 12 μM while in the second run 10 μM and 11 μM were determined. 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.

DPRA (OECD 442C)

The skin sensitisation of the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was evaluated according to OECD guideline 442C (Direct Peptide Reactivity Assay, DPRA).

At the beginning of the assay the solubility of the test chemical was assessed and acetonitrile was chosen as the appropriate solvent with which the test item formed a homogenous solution. Concentrations of the synthetic cysteine and lysine peptides following the 24 hours incubation time were determined by HPLC with gradient elution and UV detection at 220 nm.

Cysteine and lysine depletion values were to be used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptide, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used. The threshold of 6.38 % mean percent peptide depletion supported the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA).

The positive control replicates showed the expected percent peptide depletion values within acceptable limits (68.53% with cysteine peptide and 51.63 % mean percent lysine peptide depletion). The back-calculated values of the reference control replicates were within the expected molarity concentration range of 0.50 ± 0.05 mM. The experiment was considered to be valid.

The percent cysteine peptide depletion value of Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides was 3.58 % while the percent lysine peptide depletion was 1.79 %. The Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers based on the threshold of 6.38 %. The average percent peptide depletion value of the test item was 2.69 % which does not exceed the threshold. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item Ethanol, 2,2’-iminobis-, N-tallow alkyl derivs., N-oxides indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.

Conclusion 

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of this validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90 % and an accuracy of 90 %.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012; Table 3). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 3: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/h-CLAT assays

DPRA	LuSens/ KeratinoSens™	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	Negative	non-sensitizer

 

 In accordance with the published evaluation scheme (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.