Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Endpoint summary

Description of key information

There are three in-chemico/in-vitro tests available for this substance. There is a Direct Peptide Reactivity Assay (DPRA), which did not show any indication of depletion of the cysteine or lysine peptides. There is also a Keratinosens assay which did not show skin sensitisation potential. However the h-CLAT assay did show an increase above 200% for the CD54 in two out of three runs. However there was very significant variability between the runs, with one run showing no indication of increases near to the threshold for either CD54 or CD86.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th September 2016 - completed 18th January 2017 - reported 23rd February 2017

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:

Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154,
January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

ECHA Guidelines 7a July 2017, require the use of this test prior to the LLNA as animal testing is a last resort. The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP .
[4], [12]. It is therefore a crucial step for the sensitising potential of a chemical.

Details on the study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.

Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run .

Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
.
Reference control C was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Peptides
20.36 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.532 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.

21.37 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.732 mL) to reach a concentration of 0.667 mM.

All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups
Reference Control C (solvent control) undiluted
Test Item 100 mM stock solution
Positive Control 100 mM stock solution

Pre-Experiments
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). The test item was dissolved in the following solvents suitable for the test:
- acetonitrile
- water
The test item was soluble in water, which was used as solvent for the test.

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 2.

Key result

Parameter:

other: Peptide depletion %

Remarks:

Prediction model 1

Value:

0.46

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Preptide depletion %

Remarks:

Prediction Model 2

Value:

0.59

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in water. No turbidity,

precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using water.  

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples.  

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed.

Table 6 Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area  at 220 nm	Peptide Concentration [mM]	Peak Area  at 220 nm	Peptide Concentration [mM]
STD1	4655.6279	0.5340	4493.0674	0.5340
STD2	2355.5945	0.2670	2277.1133	0.2670
STD3	1163.3136	0.1335	1160.3005	0.1335
STD4	583.4137	0.0667	577.1267	0.0667
STD5	288.7250	0.0334	292.0436	0.0334
STD6	140.0172	0.0167	147.2783	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Based on these results, linear regression was performed and the calibration curves were determined.

Results of the Cysteine Peptide Depletion

Table 7: Results of the Cysteine Peptide Depletion

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1333.1064	0.1526	69.69	70.06	0.48	0.68
	1323.4985	0.1515	69.91
	1292.8408	0.1479	70.60
Test Item	4333.3467	0.4960	0.00	0.59	0.64	109.09
	4306.2905	0.4929	0.49
	4272.4668	0.4890	1.27

 

Table 8: Results of the Lysine Peptide Depletion

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1802.2493	0.2125	57.49	57.48	1.25	2.18
	1749.5433	0.2062	58.73
	1855.6707	0.2188	56.23
Test Item	4122.5361	0.4883	0.18	0.33	0.14	43.44
	4110.8330	0.4870	0.46
	4115.9360	0.4876	0.34

 

Table 9: Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed. Since precipitation of the lysine peptide was observed, prediction model 2 should be considered.

Predicition Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2  (Cysteine Peptide / TestItemRatio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.46	Minimal Reactivity	no sensitizer	0.59	Minimal Reactivity	no sensitizer
Positive Control	63.77	High Reactivity	sensitizer	70.06	High Reactivity	sensitizer

 

Table 10 Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	1.0000	pass
mean peptide concentration of RC A	0.45≤x≤0.55 mM	0.5149	pass
mean peptide concentration of RC C (PC)	0.45≤x≤0.55 mM	0.5033	pass
mean peptide concentration of RC C (TI)	0.45≤x≤0.55 mM	0.4953	pass
CV of the peak area of RC B	< 15%	1.54	pass
CV of the peak area of RC C (PC)	< 15%	0.77	pass
CV of the peak area of RC C (TI)	< 15%	1.43	pass
mean peptide depletion of the PC	60.8% < x < 100%	70.06	pass
SD of peptide depletion of the PC replicates	< 14.9%	0.48	pass
SD of peptide depletion of the TI replicates	< 14.9%	0.64	pass

 

Table 11 Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9999	pass
mean peptide concentration of RC A	0.45≤x≤0.55 mM	0.5024	pass
mean peptide concentration of RC C (PC)	0.45≤x≤0.55 mM	0.5023	pass
mean peptide concentration of RC C (TI)	0.45≤x≤0.55 mM	0.4892	pass
CV of the peak area of RC B	< 15%	0.28	pass
CV of the peak area of RC C (PC)	< 15%	0.15	pass
CV of the peak area of RC C (TI)	< 15%	0.18	pass
mean peptide depletion of the PC	40.2% < x < 69.0%	57.48	pass
SD of peptide depletion of the PC replicates	< 11.6%	1.25	pass
SD of peptide depletion of the TI replicates	< 11.6%	0.14	pass

 

Historical Data

Table 12 Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9995	0.0005	22
mean peptide concentration of reference A [mM]	0.5019	0.0000	22
mean peptide concentration of reference C [mM]	0.4873	0.0000	36
CV of the peak area of control B [%]	2.43	1.39	22
CV of the peak area of control C [%]	1.72	1.45	36
mean peptide depletion of the PC [%]	73.11	1.54	22
SD of peptide depletion of the PC replicates [%]	0.69	0.45	22
SD of peptide depletion of the test items [%]	13.02	26.77	57

 

Table 13 Historical Data Lysine Peptide

Lysine Peptide
	Mean	SD	N
linearity of the calibration curve	0.9999	0.0001	21
mean peptide concentration of reference A [mM]	0.4868	0.0157	21
mean peptide concentration of reference C [mM]	0.4868	0.1938	34
CV of the peak area of control B [%]	0.64	0.19	21
CV of the peak area of control C [%]	0.50	0.62	34
mean peptide depletion of the PC [%]	59.28	6.86	21
SD of peptide depletion of the PC replicates [%]	1.67	1.48	21
SD of peptide depletion of the test items [%]	1.62	2.33	57

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine and lysine peptide. The test item might be considered as “non-sensitizer”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach
such as IATA.

Executive summary:

Results

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing

either lysine or cysteine.  

In the present study N-[2-(2-Hydroxyethoxy)ethyl]acetamide was given into water, based on the results of the pre-experiments. Based on the weighted molecular weight of 137.26 g/mol a 100 mM

stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

The test item was completely soluble and the resulting solution was used for further testing.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation.  

For the cysteine peptide experiment no precipitation, turbidity or phase separation was observed for any test sample.

For the lysine peptide experiment no precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and

the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by

comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.46%). Based on prediction model 1 the test

item can be considered as non-sensitiser.

Since the positive control fulfilled all quality criteria the observed precipitations were considered as irrelevant. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high

reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.77%.

Conclusion

In this study under the given conditions the test item showed minimal reactivity towards the cysteine and the lysine peptide. The test item might be considered as “non-sensitizer”.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach

such as IATA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Star of study 2nd November 2016 - Final report 10th March 2017

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:

KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not
sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as an IATA, combining them with other
complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP

Details on the study design:

Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; Sigma; Lot No.: SZBG0830V, Merck, Lot: K46959552, Applichem, Lot: 0000803731). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.

These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO (Sigma; Lot No.: SZBG0830V, Merck, Lot: K46959552, Applichem, Lot: 0000803731) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Sigma-Aldrich; Lot No.: MKBS26662) was used as positive control. CA was dissolved in DMSO (Merck; Lot No.:
K46959552, Applichem, Lot: 0000803731 ) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM.
The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the
solvent DMSO was 1% (v/v) for all wells.

Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 03 experiment 1, P 05 experiment 2, P 04 experiment 3) were used.

Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +/- 1°C and 5% CO2. For test material exposure, cells were cultured in medium for test item exposure.

Composition of Media
Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885- 108, Lot No.: 1801714, 1829961) with 1.0 g/L D-glucose and Na-Pyruvate. The medium will be supplemented
with the following components:
- 10% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0391E, 0596E)
- 1% geneticin (final concentration: 500 µg/mL; Gibco Life Science, Cat. No. 10131-027, Lot No.: 1717097, 1803405)

Assay Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-108, Lot No.: 1801714, 1829961) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0391E, 0596E)

Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-108, Lot No.: 1801714, 1829961) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0391E, 0596E)

Luciferase Assay System
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacturer’s manual.

Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000211803, 0000220668) consisted of the following components relevant for this study:
- 10 x Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.

Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000199324) consists of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:4 with dist. water (Braun; Lot No.: 162218071,Sigma; Lot No.: RNBF3331)

Further Reagents
MTT solution
MTT solution
- MTT (Sigma-Aldrich, CAS No.: 298-93-1, Lot No.: MKBR6576V) stock solution: 5 mg/mL
MTT in DPBS (Gibco Life Science; Lot No.: 1813255)
SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS; AppliChem, CAS No.: 151-21-3, Lot
No.: 2N004694, 40015277) in dist. water (Sigma; Lot No.: RNBF3331)

DPBS:
DPBS solution (without Ca2+/Mg2+) (Gibco Life Science; Lot No.: 1813255)

Dose Groups
1. Negative Control: DMSO: 1% (v/v) in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates.

Experimental Procedure
A cell suspension of 8 × 10E4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10E4 cells were dispensed in each well. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.

After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.

All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of
light.

Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and OD was measured at λ = 600 nm.

Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of
discordant results a third independent run should be performed.

The following parameters were calculated:

Calculation of Cell Viability
Cell viability was calculated according to equation 1.
Cell Viability[%] = (Vsample-Vblank) x100
(Vsolvent-Vblank)

Vsample = MTT absorbance reading in the test chemical well
Vblank = MTT absorbance reading in the blank well containing no cells and no treatment
Vsolvent = average MTT absorbance reading in the wells containing cells and solvent (negative) control

Calculation of the Maximal Induction of the Luciferase Activity (Imax)
The maximal average fold induction of luciferase activity (Imax) value observed at any concentration
of the test item was calculated according to equation 2.

Fold induction = (Lsample-Lblank)
(Lsolvent-Lblank)

Lsample = luminescence reading in the test chemical well
Lblank = luminescence reading in the blank well containing no cells and no treatment
Lsolvent = luminescence reading in the wells containing cells and solvent (negative) control

Calculation of the EC1.5
The EC1.5 will be calculated by linear extrapolation according to equation 3, and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.

EC1.5 = (Cb - Ca) x (1.5-Ia) + Ca
(Ib-Ia )
Ca = lowest concentration in µM with >1.5 fold induction
Cb = highest concentration in µM with <1.5 fold induction
Ia = fold-induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib = fold-induction measured at the highest concentration with <1.5 fold induction (mean of three replicate wells)

Calculation of IC50 and IC30
The IC50 and IC30 was calculated by linear extrapolation according to equation 4 and the overall IC50 and IC30 was calculated as the geometric mean of the individual repetitions.

ICx = (Cb - Ca) x ( (100-x))-Va) + Ca
( Vb-Va )

X = % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca = the lowest concentration in µM with >X% reduction in cell viability
Cb = highest concentration in µM with Va = % viability at the lowest concentration with >X% reduction in cell viability
Vb = % viability at the highest concentration with
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.

The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction on cellular viability at the EC1.5 determining concentration.

Prediction Model:
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions.


- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative
control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 1000 µg/mL
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition will be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM were considered as inconclusive.

Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition consisting of 6 wells.

Key result

Run / experiment:

other: Based on Experiment 1 and 2

Parameter:

other: Luciferase activity increase - No relevant dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Cytotoxicity

Table 1: Results of the Cytotoxicity Measurement  

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	100	100	100	100	0.0
Positive Control	4.00	105.6	104.5	107.8	106.0	1.7
	8.00	109.3	103.7	106.6	106.5	2.8
	16.00	113.3	114.1	118.6	115.3	2.8
	32.00	120.4	127.3	123.2	123.6	3.5
	64.00	119.8	120.7	129.4	123.3	5.3
Test Item	0.98	84.3	127.0	91.0	100.8	23.0
	1.95	91.5	89.2	88.8	89.8	1.5
	3.91	84.8	116.9	96.5	99.4	16.2
	7.81	87.4	124.3	89.6	100.4	20.7
	15.63	88.1	121.3	99.3	102.9	16.9
	31.25	80.6	111.9	99.9	97.5	15.8
	62.50	82.0	119.6	101.3	101.0	18.8
	125.00	81.8	114.1	109.9	101.9	17.6
	250.00	81.7	117.6	108.5	102.6	18.6
	500.00	81.6	110.4	108.3	100.1	16.0
	1000.00	79.3	102.9	98.7	93.6	12.6
	2000.00	84.2	101.1	109.4	98.2	12.8

 

Luciferase Activity

Table 2: Induction of Luciferase Activity Experiment 1

	Concentration [µM]	Cell Viability [%]					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.28	1.42	1.18	1.29	0.13
	8.00	1.34	1.21	1.18	1.24	0.09
	16.00	1.86	2.01	1.83	1.90	0.10	*
	32.00	2.16	1.79	2.23	2.06	0.24	*
	64.00	3.94	4.02	3.95	3.97	0.04	*
Test Item	0.98	0.97	1.71	1.34	1.34	0.37
	1.95	0.82	1.54	1.34	1.23	0.37
	3.91	0.77	1.55	1.30	1.21	0.40
	7.81	0.86	1.46	1.45	1.26	0.34
	15.63	0.84	1.64	1.21	1.23	0.40
	31.25	0.86	1.50	1.13	1.16	0.34
	62.50	0.94	1.49	1.16	1.20	0.28
	125.00	0.94	1.79	1.04	1.26	0.46
	250.00	1.21	2.37	1.25	1.61	0.66
	500.00	0.83	1.69	1.14	1.22	0.44
	1000.00	0.94	1.53	1.29	1.25	0.29
	2000.00	0.88	1.58	1.02	1.16	0.37

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Table 3: Induction of Luciferase Activity Experiment 2

	Concentration [µM]	Cell Viability [%]					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.26	1.00	1.23	1.17	0.14
	8.00	1.21	1.13	1.08	1.14	0.07
	16.00	1.48	1.11	1.53	1.37	0.23
	32.00	1.74	1.46	1.55	1.58	0.15	*
	64.00	2.93	2.24	3.02	2.73	0.43	*
Test Item	0.98	1.32	0.91	1.05	1.09	0.21
	1.95	1.03	0.87	0.94	0.95	0.08
	3.91	0.99	0.90	1.02	0.97	0.06
	7.81	1.00	0.80	1.02	0.94	0.12
	15.63	1.02	0.90	0.99	0.97	0.06
	31.25	1.01	0.83	0.93	0.92	0.09
	62.50	0.96	0.83	0.91	0.90	0.06
	125.00	0.96	0.89	0.97	0.94	0.05
	250.00	0.96	0.80	0.92	0.90	0.09
	500.00	0.95	0.92	0.96	0.94	0.02
	1000.00	0.92	0.76	0.98	0.89	0.11
	2000.00	1.02	0.77	1.02	0.94	0.14	*

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity Experiment 3

	Concentration [µM]	Cell Viability [%]					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.09	1.40	0.59	1.03	0.41
	8.00	1.28	1.76	0.91	1.31	0.43
	16.00	1.52	1.98	1.24	1.58	0.37
	32.00	2.29	2.72	1.76	2.26	0.48	*
	64.00	3.45	6.01	5.38	4.95	1.34	*
Test Item	0.98	0.97	1.36	2.33	1.55	0.70
	1.95	1.11	1.34	1.04	1.16	0.16
	3.91	1.02	1.29	1.40	1.24	0.20
	7.81	1.12	1.37	1.29	1.26	0.13
	15.63	1.09	1.14	1.45	1.22	0.20
	31.25	1.04	1.11	0.87	1.01	0.12
	62.50	1.69	1.15	1.00	1.28	0.36
	125.00	1.20	1.22	0.89	1.11	0.18
	250.00	1.31	1.11	1.00	1.14	0.15
	500.00	0.95	1.24	0.75	0.98	0.25
	1000.00	0.95	1.25	0.90	1.03	0.19
	2000.00	1.00	0.99	0.80	0.93	0.11

* = significant induction according to Student’s t-test, p<0.05

Luciferase Activity - Overall Induction

Table 5: Induction of Luciferase Activity – Overall Induction

 

	Concentration [µM]	Cell Viability [%]					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.29	1.17	1.03	1.16	0.13
	8.00	1.24	1.14	1.31	1.23	0.09
	16.00	1.90	1.37	1.58	1.62	0.27	*
	32.00	2.06	1.58	2.26	1.96	0.35	*
	64.00	3.97	2.73	4.95	3.88	1.11	*
Test Item	0.98	1.34	1.09	1.55	1.33	0.23
	1.95	1.23	0.95	1.16	1.11	0.15
	3.91	1.21	0.97	1.24	1.14	0.15
	7.81	1.26	0.94	1.26	1.15	0.18
	15.63	1.23	0.97	1.22	1.14	0.15
	31.25	1.16	0.92	1.01	1.03	0.12
	62.50	1.20	0.90	1.28	1.13	0.20
	125.00	1.26	0.94	1.11	1.10	0.16
	250.00	1.61	0.90	1.14	1.22	0.37
	500.00	1.22	0.94	0.98	1.05	0.15
	1000.00	1.25	0.89	1.03	1.06	0.19
	2000.00	1.16	0.94	0.93	1.01	0.13

* = significant induction according to Student’s t-test, p<0.05

Additional Parameters

Table 6: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5 [µM]	209.68	n.a.	n.a.	n.a.	n.a.
Imax	1.61	1.09	1.55	1.42	0.28
IC30 [µM]	n.a.	n.a.	n.a.	n.a.	n.a.
IC50 [µM]	n.a.	n.a.	n.a.	n.a.	n.a.

 

Acceptance Criteria

Table 7: Acceptance Criteria

Criterion	Range	Exp. 1	pass/fai	Exp. 2	pass/fai	Exp. 3	pass/fai
CV Solvent Control	< 20%	15.2	pass	9.6	pass	12.3	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	2.0	pass	3.0	pass
EC1.5 PC	7 < x < 30 µM	11.12	pass	25.59	pass	13.57	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.97	pass	2.73	pass	4.95	pass

 

Historical Data

Table 8: Historical Data

Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.7	3.1	39
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥1	2.4	0.5	39
EC1.5 PC	7 < x < 30 µM	18.29	5.47	39
Induction PC at 64 µM	2.00 < x < 8.00	3.52	1.02	39

 

Interpretation of results:

GHS criteria not met

Conclusions:

Conclusion

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

Results

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.  

In the present study N-[2-(2-Hydroxyethoxy)ethyl]acetamide was dissolved in DMSO.

Based on a molecular weight of 147.17 g/mol a stock solution of 200 mM was prepared.  

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.61 was determined at a test item concentration of 250.00 µM. The corresponding cell viability was 81.7%. The calculated EC1.5 was < 1000 µM (209.68 µM).

In the second experiment, a max luciferase activity (Imax) induction of 1.09 was determined at a test item concentration of 0.89 µM. The corresponding cell viability was 127.0%. No EC1.5 could be calculated.

In the third experiment, a max luciferase activity (Imax) induction of 1.55 was determined at a test item concentration of 0.98 µM. The corresponding cell viability was 91.0%. no EC1.5 could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Conclusion

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study started 24 October 2016 - 4th April 2017 - Final Report 9th August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st,
2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

Details on the study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%, Lot No.: 0000803731) at a concentration of 500 mg/mL, respectively. Test items of limited solubility were dissolved at the highest soluble concentration in DMSO. Stock solutions were prepared diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.

The working stock solutions were prepared diluting each stock solution 250 times with cell culture medium.

The working stock solutions were applied to the cells by adding equal volumes of each solutions to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
Solvent controls and a positive control were set up in parallel in order to confirm the validity of the test.

Solvent Controls
The solvent control was set up depending on the appropriate solvent previously determined (see chapter 10.6.2).

For chemicals solubilized in either cell culture medium or 0.9% NaCl, cell culture medium was the solvent control.

For chemicals solubilized in DMSO, DMSO will be the solvent control.

The solvent controls will be diluted according to the procedure described for the test item in the Experimental proceedures chapter, resulting in a final concentration of 1% (v/v) for 0.9% NaCl and 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB; CAS No.: 97-00-7, purity ≥ 99 %, Lot No.: BCBP8259V) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described
for the test item in experimental proceedures chapter, resulting in a final DMSO concentration of 0.2% (v/v).

Test System
FACS
FACS: BD Canto II
Software BD FACS DIVA 6.0

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used.

Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum (FBS, FBS, Biochrome, Cat No.: S 0615, Lot: 0391E), 25 mM HEPES (Gibco Life Science, Cat No.:15630-056 Lot.: 1788128), L-glutamine (Gibco Life Science, Cat. No.: 25030-081, Lot: 1801687), 2-mercaptoethanol (Gibco Life Science, Cat No.: 21985-023, Lot: 1779219) and penicillin/streptomycin (Gibco Life Science, Cat. No.: 15240-062, Lot: 1796438) at 37 +/- 1°C and 5% CO2.

Dose Groups
1. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
2. Positive Control: 4 µg/mL DNCB
3. Test Item: 8 concentrations of the test item (dose finding assay/ main experiment)

dose finding assay 1 and 2:
1000, 500, 250, 125, 62.50, 31.25, 15.63 and 7.81 µg/mL

main experiment 1 and 2:
60.63; 50.53; 42.10, 35.09; 29.24; 24.37; 20.30; 16.92 µg/mL

Pre-Experiments
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB (CAS No.: 97-00-7, ≥99% purity,
Aldrich, Lot No.: BCBP8259V) at a final concentration of 4 µg/mL and nickel sulphate (CAS No.: 10101-97-0, ≥99% purity, Carl Roth, Lot No.: 384216612) at a final concentration of 100 µg/mL served as positive control while lactic acid (CAS No.: 50-21-5, ≥99% purity, Fluka, Lot No.: BCBQ0345V) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl (Braun; Lot No.: 160948002) at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. Test items
not soluble in DMSO at 500 mg/mL, were solved at the highest soluble concentration in DMSO by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL. The test item was tested in the most applicable solvent at its highest concentration.

Experimental Procedure

Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally
used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution .

For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well).

The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

After 24 h ± 1 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e.
FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.

The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an exication wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells will be acquired and cell viability
will be calculated for each test concentration according to equation 10-1.

The CV75 value, i.e. the concentration showing 75% cell survival, will be calculated by log-linear interpolation utilizing equation 10-2. The CV75 value will be used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was solved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2;
CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.

The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions will be further diluted 250-fold into the culture medium (working
solutions). These working solutions are finally used for cell treatment with a further final 2-fold dilution factor. Alternative concentrations were used upon justification (e.g. in case of poor solubility or cytotoxicity)

For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

After 24 h ± 1 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained
with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).

The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm or FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated according to equation 10-3. The cell viability was calculated according to equation 10-1.

Data Analysis
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The
mean values and standard deviations of the single replicates were determined using the respective excel commands.

Calculation of Cell Viability 10.1

Cell Viability = Number of living cells x 100
Total number of aquired Cells

Calculation of CV75 - 10-2

Log CV75 = (75-c)*Log b-(75 -a)* Log d
a-c
a = the lowest cell viability over the threshold of 75% cell viability
b = the concentration of the test item, which causes a
c = the highest cell viability under the threshold of 75% cell viability
d = the concentration of the test item, which causes c

Calculation of the Realtive Fluoescence Intensity (RFI) - 10-3

RFI =MFI test item treated cells - MFI test item treated isotype cell
MFI solvent treated calls - MFI solvent treated isotype cells

RFI = relative fluorescence intensity
MFI = geometric mean fluorescence intensity

Evaluation of the Results

Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent
runs to derive a single prediction. Each independent run was performed on a different day or on the
same day provided that for each run: independent fresh stock solutions and working solutions of the
test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86
and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was
equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two
independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a
cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54
were equal to or are greater than 150% and 200% respectively at any tested dose at a cell
viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should
be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x
CV75 is <90%. In contrast, a positive test outcome will be accepted irrespective of cell viabilities
>90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be
accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9%
NaCl solution; 1000 µg/mL for DMSO) even if the cell viability is >90%.

Effective Concentration 150 and Effective Concentration 200 values:
For test chemicals classified as sensitiser the effective concentration 150 for CD86 (EC150) and the effective concentration 200 for CD54 (EC200) can be calculated with following formula 10-4:

EC150 =Bdose +[( 150-BRFI)/(ARFI-BRFI)* (Adose -Bdose)]
EC200 =Bdose +[( 200-BRFI)/(ARFI-BRFI)* (Adose -Bdose)]

If the RFI value of the lowest dose is above the positive criteria of CD86 and CD54, EC150 and EC300 values can be calculated using the lowest dose by log linear extrapolation according to the following formula 10-5:

EC150 =2{log2(Bdose)-(150-BRFI)/(ARFI-BRFI)*[log2 (Adose -Bdose)]}
EC200 =2{log2(Bdose)-(200-BRFI)/(ARFI-BRFI)*[log2 (Adose -Bdose)]}

Adose is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
Bdose is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)


For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs should be performed for CD86/CD54 expression measurement. The EC150 and EC200 values are the median value of the ECs calculated from three independent runs. In order to select the median
value, three independent runs are necessary. If only two of three independent runs meet the positive criteria, the higher EC150 or EC200 of the two calculated values is adopted. The EC values could potentially contribute to the assessment of sensitising potency [8] when used in integrated
approaches such as IATA [4].

Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

Key result

Run / experiment:

other: Experiment 1 and 3

Parameter:

other: Upregulation of CD54 in two independent experiment runs

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 42.7 h which is within the doubling time range specified by the manufacturer (35 - 50 h).

Table 1: Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86		CD54		Activated
		Cell viability [%]	RFI	Cell viability [%]	RFI	yes/no
DNCB	4	88.1	406	88.4	243	Yes
NiSO4	100	88.4	399	87.9	450	Yes
LA	1000	96.7	85	96.7	103	No

 

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and

CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in 0.9% NaCl solution at a concentration of 100 mg/mL and up to 500 mg/mL.  

Dose Finding Assay

Three dose finding assays were performed. The first and second dose finding assays were performed using stock solutions with a concentration of 100 mg/mL in 0.9% NaCl. Since the final

highest concentration of 1000 µg/mL was non-toxic, the maximum concentration was re-determined by performing a new cytotoxicity test, up to a final concentration of 5000 µg/mL. Therefore, the third dose finding assay was performed using stock solutions with a concentration of 500 mg/mL.

Table 2: Results of the Dose Finding Assay

Sample	Experiment 1		Experiment 2		Experiment 3
	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	0.00	98.80	0.00	98.30	0.00	97.90
DMSO Control	4.00	98.80	0.00	98.30	0.00	97.90
N-[2-(2-Hydroxyethoxy) ethyl]acetamide	7.81	98.80	7.81	98.50	39.06	97.50
	15.63	98.60	15.63	98.30	78.13	98.10
	31.25	98.90	31.25	98.10	156.25	97.80
	62.50	98.70	62.50	98.30	312.50	97.80
	125.00	98.90	125.00	98.40	625.00	97.70
	250.00	98.70	250.00	98.40	1250.00	97.70
	500.00	98.70	500.00	98.30	2500.00	97.30
	1000.00	98.90	1000.00	98.60	5000.00	96.80
Calculated CV75 [µg/mL]	No CV75		No CV75		No CV75
Mean CV75 [µg/mL]	No CV75
SD CV 75 [µg/mL]	-

Three single runs were performed in order to determine the mean CV75. No CV75 could be calculated, since cell viability was >75% up to the highest concentration tested. Based on this, the main experiment was performed covering a concentration range from 5000 – 1395 µg/mL (500.00 – 139.5 mg/mL stock solution).

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 three independent experiments were performed using separate cultivated cells at passage 12 (first experiment), 13 (second experiment) and 17 (third experiment). For each experiment separately weighted samples and preparations were used.

Table 3: CD54 and CD86 Expression Experiment1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD 86	CD 54	Isotype IgG1	CD 86	CD 54	Isotype   IgG1	CD86	CD 54	CD 86	CD 54	CD 86	CD 54
Medium Control	-	96.7	97.1	97.4	1452	965	824	628	141	100	100	176	117
DMSO Control	0.20%	96.3	96.4	95.7	1659	1032	809	850	223	135	158	205	128
DNCB	4.00	86.0	86.7	86.4	3400	1857	831	2569	1026	302	460	409	223
N-[2-(2- Hydroxyethoxy) ethyl]acetamide	5000.00	96.1	96.3	96.5	1344	1012	656	688	356	110	252	205	154
	4166.67	96.1	96.3	96.3	1615	1174	769	846	405	135	287	210	153
	3472.22	95.6	95.0	95.9	1560	964	690	870	274	139	194	226	140
	2893.52	95.7	95.5	95.8	1464	916	647	817	269	130	191	226	142
	2411.27	95.6	95.5	95.7	1411	945	720	691	225	110	160	196	131
	2009.39	95.3	95.2	95.7	1322	885	753	569	132	91	94	176	118
	1674.49	95.6	95.4	95.8	1395	868	697	698	171	111	121	200	125
	1395.41	96.0	95.4	96.3	1307	861	680	627	181	100	128	192	127

 

  Table 4: CD54 and CD86 Expression Experiment2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD 86	CD 54	Isotype IgG1	CD 86	CD 54	Isotype   IgG1	CD86	CD 54	CD 86	CD 54	CD 86	CD 54
Medium Control	-	96.2	96.1	96.3	2007	1584	972	1035	612	100	100	206	163
DMSO Control	0.20%	95.9	96.3	95.7	2323	1606	954	1369	652	132	107	244	168
DNCB	4.00	88.3	88.7	88.7	6095	3811	955	5140	2856	375	438	638	399
N-[2-(2- Hydroxyethoxy) ethyl]acetamide	5000.00	97.0	96.5	96.5	2025	1551	791	1234	760	119	124	256	198
	4166.67	96.1	95.7	95.7	1998	1534	829	1169	705	113	115	241	185
	3472.22	96.5	96.5	96.1	1972	1518	822	1150	696	111	114	240	185
	2893.52	96.1	96.5	96.3	2059	1427	852	1207	575	117	94	242	167
	2411.27	96.3	96.1	96.1	2062	1535	900	1162	635	112	104	229	171
	2009.39	96.1	96.1	95.8	2087	1463	890	1197	573	116	94	234	164
	1674.49	96.0	96.4	96.3	1998	1439	890	1108	549	107	90	224	162
	1395.41	96.1	95.8	95.7	1999	1472	957	1042	515	101	84	209	154

Table 5: CD54 and CD86 Expression Experiment3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD 86	CD 54	Isotype IgG1	CD 86	CD 54	Isotype   IgG1	CD86	CD 54	CD 86	CD 54	CD 86	CD 54
Medium Control	-	96.9	97.0	97.0	1314	1093	896	418	197	100	100	147	122
DMSO Control	0.20%	96.7	96.7	96.4	1351	1124	826	525	298	126	151	164	136
DNCB	4.00	86.1	87.4	86.3	2870	2341	789	2081	1552	396	521	364	297
N-[2-(2- H1117 ydroxyethoxy) ethyl]acetamide	5000.00	96.3	96.4	96.4	1423	1187	726	697	461	167	234	196	163
	4166.67	96.4	96.4	96.7	1442	1126	756	686	370	164	188	191	149
	3472.22	96.1	96.3	96.4	1416	1117	761	655	356	157	181	186	147
	2893.52	96.3	95.8	96.2	1499	1104	795	704	309	168	157	189	138
	2411.27	96.8	96.8	96.7	1413	1100	812	601	288	144	146	174	135
	2009.39	96.3	96.5	96.3	1405	1075	811	594	264	142	134	173	133
	1674.49	96.5	96.7	96.7	1394	1084	851	543	233	130	118	164	127
	1395.41	96.6	96.6	96.2	1381	1108	853	528	255	126	129	162	130

 

Table 6: Acceptance Criteria

 

Acceptance Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail	Experiment 3	pass/fail
cell viability solvent controls [%]	>90	95.7 - 97.4	pass	95.7 - 96.3	pass	95.7 - 96.3	pass
number of test dosed with viability >50% CD86	>4	8	pass	8	pass	8	pass
number of test dosed with viability >50% CD54	>4	8	pass	8	pass	8	pass
number of test dosed with viability >50% IgG1	>4	8	pass	8	pass	8	pass
RFI of positive control of CD86	≥150	302	pass	375	pass	396	pass
RFI of positive control of CD54	≥200	460	pass	438	pass	521	pass
RFI of solvent control of CD86	<150	135	pass	132	pass	126	pass
RFI of solvent control of CD54	<200	158	pass	107	pass	151	pass
MFI ratio IgG1/CD86 for medium control [%]	>105	176	pass	206	pass	147	pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	205	pass	244	pass	164	pass
MFI ratio IgG1/CD54 for medium control [%]	>105	117	pass	163	pass	122	pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	128	pass	168	pass	136	pass

 

Table 6: Historical Data

Criterion	Mean	SD	N
Cell viability solvent controls [%]	97.1	21.8	442
number of test dosed with viability >50%	-	-	1060
RFI of positive control of CD86	408.9	173.5	73
RFI of positive control of CD54	655.4	331.5	73
RFI of solvent control of CD86	116.9	14.6	72
RFI of solvent control of CD54	124.8	26.6	72
MFI ratio IgG1/CD86 for medium control [%]	193.1	49.2	74
MFI ratio IgG1/CD86 for DMSO control [%]	213.0	60.9	74
MFI ratio IgG1/CD54 for medium control [%]	129.8	13.2	74

 

Interpretation of results:

study cannot be used for classification

Remarks:

Study is positive, but is not not sufficient on its own to classify as a skin sensitiser.

Conclusions:

IConclusion

In this study under the given conditions the test item did upregulate one of the cell surface marker in at least two independent experiment runs. Therefore, the test item indicates skin sensitizing potential in accordance with UN GHS category 1.

There are currently three in-chemico / In-vitro skin sensitisation test methods specified in ECHA guidance, the other two tests for this substance, the DPRA and the Keratinosens were negative. Based on this one approach that has been proposed is to use a two or more out of three positive responses to trigger skin sensitisation classification, in which case Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine could be considered not to require classification as a skin sensitiser. However the current ECHA guidance does not endorse this approach. Also the current ECHA guidance requires for substances classified as skin sensitisers that we classify as 1A or 1B, in-vitro results can only result in classification as category 1A. Based on this equivocal result with only one positive result in the h-CLAT, the available information is considered not to be sufficient for classification. Therefore an animal test is necessary to confirm if this substance should be classified as a skin sensitiser and if so which category.

Executive summary:

Summary

Results

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP),

namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared

to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

 

In the present study N-[2-(2-Hydroxyethoxy)ethyl]acetamide was dissolved in 0.9% NaCl. For the first and second dose finding assays stock solutions with a concentration from 0.78 to 100 mg/mL

were prepared by a serial dilution of 1:2. For the third dose finding assay stock solutions with a concentration from 3.9 to 500 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated

with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 could be calculated, since viability was <75% up

to the highest test item concentration.

Based on this, the main experiment was performed covering the following concentration steps:

5000; 4167; 3472, 2894; 2411; 2009; 1674 and 1395 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in

parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 96.1% (CD86), 96.3% (CD54) and 96.5% (isotype

IgG1 control) in the first experiment, to 97.0% (CD86), 96.5% (CD54) and 96.5% (isotype IgG1 control) in the second experiment and to 96.3% (CD86), 96.4% (CD54) and 96.4% (isotype IgG1

control) in the third experiment.

In the first experiment the expression of the cell surface marker CD54 was upregulated to 252%. The upregulation above the threshold of 200% was observed starting from a concentration of

4167 µg/mL. In contrast, the expression of cell surface marker CD86 was not upregulated above the threshold of 150% in this first experiment.

In the second experiment the expression of both the cell surface marker (CD86 and CD54) was not upregulated above the respective thresholds. In the third experiment the expression of the cell surface marker CD86 was upregulated to 168%. The upregulation above the threshold of 150% was observed starting from a concentration of 2894 µg/mL. The expression of the cell surface marker CD54 was upregulated to 234%. The upregulation above the threshold of 200% was observed at the highest concentration of 5000 µg/mL.

Since one of the cell surface marker (CD54) clearly exceeded the threshold in two independent experiments the test item indicates skin sensitizing potential. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (302% experiment 1; 375% experiment 2; 396% experiment 3) and 200% for CD54 (460% experiment 1; 438% experiment 2; 521% experiment 3) were clearly exceeded.

Conclusion

In this study under the given conditions the test item did upregulate one of the cell surface marker in at least two independent experiment runs. Therefore, the test item indicates skin sensitizing potential in accordance with UN GHS category 1.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In QSAR Toolbox (v.4.3) none of the general mechanistic and endpoint specific profilers that are relevant for skin sensitization triggered a concern to the DGA structure.

General mechanistic

·        Protein binding by OASIS: No alert found

·        Protein binding by OECD: No alert found

·        Protein binding potency GSH: Not possible to classify according to these rules (GSH)

·        Protein binding potency Cys (DPRA 13%): DPRA less than 9% (DPRA 13%)

·        Protein binding potency Lys (DPRA 13%): DPRA less than 9% (DPRA 13%)

Endpoint specific

·        Keratinocyte gene expression: Not possible to classify according to these rules

·        Protein binding alerts for Chromosomal aberration by OASIS: No alert found

·        Protein binding alerts for skin sensitization according to GHS: No alert found

·        Protein binding alerts for skin sensitization by OASIS: No alert found

·        Protein Binding Potency h-CLAT: No alert found

·        Respiratory sensitisation: No alert found

 

Of the relevant QSARs, TOPKAT and specially DEREK do not predict a concern for sensitization (both high reliability and no misclassified or unclassified features), and only one of the two VEGA models predict possible skin sensitization. However, this VEGA model prediction is not reliable as the DGA structure is outside the applicability domain of the model.

All in all, there are no concerns for skin sensitization. The Read-across analysis executed via the automated work process for "Skin sensitization" in QSAR Toolbox results to a negative prediction. (The prediction is based on 4 values, all negative).

 

 

There are three in-chemico/in-vitro tests available for this substance. There is a Direct Peptide Reactivity Assay (DPRA), which did not show any indication of depletion of the cysteine or lysine peptides. There is also a Keratinosens® assay which did not show skin sensitisation potential. However, the h-CLAT assay did show an increase above 200% for the CD54 in two out of three runs. However, there was very significant variability between the runs, with one run showing no indication of increases near to the threshold for either CD54 or CD86.

 

The current ECHA guidance on skin sensitisation in Guidance document 7a of July 2017 (ECHA 2017), describes the new none animal test methods to identify skin sensitisation potential. The main studies with OECD guidelines are the DPRA (The Direct Peptide Reactivity Assay) OECD TG 442C which represents KE (Key Event 1 in the AOP (Adverse Outcome Pathway) for skin sensitisation which is the MIE (molecular initiating event) as small molecules have to bind with proteins in the skin before the immune system can identify them as foreign. The Keratinosens assay OECD TG 442D, which represents the KE2 in the AOP indicates that the keratinocytes have activation of the Keap1-Nrf2-ARE pathway in response to test substances with skin sensitisation potential. The third test is the h-CLAT assay OECD TG 442E in which the activation of the dendritic cells KE3 in the AOP is identified by increases in the cell surface markers CD86 and CD54. It is possible to have false negative results in the DPRA if the test substance itself is does not bind to protein but is either a pro hapten i.e. need metabolism in the skin to the reactive molecule or a pre-hapten where oxidation is required to activate it. However, the Keratinosens cells will have some metabolic capacity so a negative result is indicative of a lack of skin sensitising potential. The h-CLAT assay is a complex and difficult assay to obtain consistent results (Kreiling et. al.2017). In this case positive responses in CD54 where only seen at 5000 µg/ml and 4166 µg/ml while in a third run there was no positive response from either marker. Also CD86 showed no response in two runs and then a response in the third at similar high concentrations. The standard upper limit concentration in the h-CLAT is 1000 µg/ml or lower depending on the cytotoxicity.  However initial dose ranging up to this level twice failed to find cytotoxicity. In this unusual situation the OECD guideline states that concentrations up to 5000 µg/ml should be tested. Again, no cytotoxicity was seen so this concentration was used for the main experiments. The two runs that gave a consistent positive response, for CD54 i.e. > 200% of the control value, only showed this at the highest concentrations well above the normal 1000 µg/ml upper concentration limit. 

 

The current ECHA Guidance does not provide any overall prediction model as to how to combine the data from the three main non-animal test methods as detailed for this substance.  Various prediction models have been proposed but the proposal of (Bauch et. al.2012) where there is a requirement for at least two out of the three tests to be positive for a test substance to be considered a skin sensitiser has gained some acceptance as a pragmatic approach to removing false positives responses based on a single positive response. Recently this approach has been found, for a group of 9 difficult substances, which are false positive in the local lymph node assay, (Kreiling et.al.2017) to provide a much lower rate of false positive results. The use of the two out of three prediction model reduced the number of false positive results and identified correctly the true positive substance tested and four true negatives.  There is also a weight of evidence argument concerning the result from the non-animal testing of N-[2-(2-hydroxyethyl)ethyl]acteamide. The negative result in the DPRA assay cannot be considered to be conclusive negative as the QSAR modelling indicates a possibility of metabolism to a protein reactive metabolite. As there is no metabolic capacity in the DPRA it would not be able to detect such a metabolite. The Keratinosens and the h-CLAT being cell based assays will have some metabolic competence but cannot be considered to have the full metabolism that would be seen in human the skin. If the AOP for skin sensitisation is followed it would be expected that a true skin sensitising chemical would show a positive response in both KE2 (Keratinosens) and KE3 (h-CLAT). The Keratinosens assay was tested up to the maximum concentration of 2000µm as per the OECD guideline, there was no cytotoxicity at this level. Based on this the study is a valid negative. The h-CLAT is normally carried out at a maximum concentration of 1000µg/ml, at this concentration the CD54 and CD86 would be well below the threshold for a positive response. So at this concentration N-[2-(2-hydroxyethyl)ethyl]acteamide would have been negative it is only its lack of cytotoxicity that allowed such an extremely high concentration to be tested and only at the highest concentrations that the positive responses in CD54 was seen.

 

The weight of evidence suggests that even if there is some potential for metabolism to a protein reactive metabolite and this was the reason for the positive response in the h-CLAT it is very weak so only seen at extremely high concentrations. Based on this it is extremely unlikely that it would meet the requirement for classification as a skin sensitiser. In the case of an equivocal result, the ECHA guidance does have the option to do an animal test the Local Lymph Node (LLNA) assay in mice as a last resort. However, as the weight of evidence does not indicate a profile that indicates a requirement to classify as a skin sensitiser and this substance is a cosmetic ingredient, an additional reason to avoid animal testing for REACH where possible, it is not proposed to do the LLNA for the REACH registration.

 

References:

- Reinhard Kreiling , Helge Gehrke, Thomas H. Broschard, Birte Dreeßen, Dorothea Eigler, David Hart, Veronika Höpflinger, Marcus Kleber, Joanna Kupny, Qiang Li, Peter Ungeheuer and, Ursula G. Sauer.In chemico, in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds. Regulatory Toxicology and Pharmacology 90 (2017) 262-276.

- ECHA (2017) European Chemicals Agency Guidance on Information Requirements and Chemical Safety Assessment.Chapter R.7a: Endpoint specific guidance, version 6.0: ECHA-17-G-18-EN, July 2017

- Bauch, C., Kolle, S.N., Ramirez, T., Fabian, E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulatory. Toxicology and. Pharmacology (2012). 63, 489-504.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The weight of evidence indicates that Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol is to be regarded as non-sensitising and, consequently, does not need to be classified for sensitisation.