Sodium (2-butoxyethoxy)acetate

Endpoint summary

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) and does neither activate keratinocytes (LuSens, OECD TG 422D) nor dendritic cells (h-CLAT, OECD TG 442E). Therefore, the substance is not predicted to be a skin sensitizer.

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:

16 January 2019 - 21 March 2019

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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Synthetic peptides:
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol), Batch No.:150219HS-MHe
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol), Batch No.: 020517HS_MHEW0119-2

Instruments and Devices:
Analytical and precision scales
Volumetric measurement tools
pH-meter
Standard laboratory glassware
Incubation chamber

Chemicals:
Water for chromatography
H2O, HPLC grade
Demineralised water
Acetonitrile for chromatography
CH3CN, ACN, HPLC grade
Trifluoroacetic acid
TFA, for spectroscopy, Merck
Isopropanol
CH3CHOHCH3, p.a.

Buffers:
Phosphate buffer pH 7.5 ± 0.05
18 (v/v) % 0.1 M sodium phosphate monobasic (of Sodium Phosphate Monobasic Monohydrate (NaH2PO4 • H2O) in purified water)
82 (v/v) % 0.1 M sodium phosphate dibasic (of Sodium Phosphate Dibasic Heptahydrate (Na2HPO4 • 7H2O) in purified water) pH was adjusted with either the monobasic or dibasic solution
Ammonium acetate buffer pH 10.2 ± 0.05
0.1 M Ammonium Acetate (CH3CO2NH4) in purified water
pH was adjusted with Ammonium Hydroxide (NH4OH) drop by drop

Positive control:
Cinnamaldehyde (CAS 104-55-2, 99.5 %,100 mM solution in acetonitrile for the cysteine peptide
100 mM solutions of the positive control chemical in acetonitrile were prepared just before use.

Peptide stock solutions
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.01085 g ± 10%). The previously calculated amount of the peptide (0.01153 g) was pre-weighted and 21.25 mL of pH 7.5 phosphate buffer was added right before beginning the assay in the valid run.
Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.01065 g ± 10%). The previously calculated amount of the peptide (0.01003 g) was pre-weighted and 18.83 mL of pH 10.2 acetate buffer was added right before beginning of the assay in the valid run.

Test item stock solution:
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0991 g ± 10 %) and weighted based on the single average molecular weight. 0.0993 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.0991 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the valid runs.

Test item samples:
Samples are prepared in triplicate for each peptide. The Cys-peptide samples are prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described. A final volume of 1 mL per sample is prepared for each sample

Incubation:
The positive control, solvent control and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h. When the test item stock solution was combined with the cysteine and lysine stock solutions (reaction samples) or the phosphate and ammonium acetate buffer (co-elution controls), the samples appeared to be clear and homogenous.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Acceptance criteria
-the standard calibration curve should have an r2 > 0.99
-the mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be ≤ 15.0%.
-the mean percent peptide depletion value of the three replicates for the positive control cinnamaldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion

The following criteria should be met for a test chemical’s results to be considered valid:
-the maximum standard deviation for the test chemical replicates should be less than 14.9 % for the percent cysteine depletion and less than 11.6% for the percent lysine depletion
-the mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 70.60 % and a mean lysine peptide depletion value of 53.00 %. The standard deviation of the percent peptide depletions of the positive control was 1.05 % and 0.49 % for the cysteine and lysine depletion, respectively. Thus, this validity criterion was also met.

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

2.44

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
Co-elution:
The test chemical did not absorb at 220 nm significantly (<10 % compared to the respective reference control) when tested with the cysteine and lysine peptides. The range of retention time for cysteine peptide was between 8.383 and 8.480 and the range of retention time for lysine peptide was between 6.118 and 6.317.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

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

Table 1: Cysteine peptide depletion values for the positive control and the test item

Sample	Peptide conc. calculated (mM)	Mean peptide conc. (mM)	Peptide depletion (%)	Standard deviation (SD) for peptide depletion (%)
ref C upw, rep I	0.50	0.49	-	-
ref C upw, rep II	0.49		-
ref C upw, rep III	0.49		-
CINNAMALDEHYDE, rep I	0.14	0.14	71.81%	1.05%
CINNAMALDEHYDE, rep II	0.15		70.09%
CINNAMALDEHYDE, rep III	0.15		69.91%
sodium (2-butoxyethoxy) acetate,  rep I	0.48	0.47	4.41%	1.72%
sodium (2-butoxyethoxy) acetate, rep II	0.48		1.57%
sodium (2-butoxyethoxy) acetate, rep III	0.47		4.69%

(upw = ultrapure water)

Table 2: Lysine peptide depletion values for the positive control and the test item

Sample	Peptide conc. calculated (mM)	Mean peptide conc. (mM)	Peptide depletion (%)	Standard deviation (SD) for peptide depletion (%)
ref C upw, rep I	0.49	0.49	-	-
ref C upw, rep II	0.49		-
ref C upw, rep III	0.49		-
CINNAMALDEHYDE, rep I	0.23	0.23	53.35%	0.49%
CINNAMALDEHYDE, rep II	0.24		52.44%
CINNAMALDEHYDE, rep III	0.23		53.22%
sodium (2-butoxyethoxy) acetate,  rep I	0.49	0.49	1.17%	0.19%
sodium (2-butoxyethoxy) acetate, rep II	0.49		1.53%
sodium (2-butoxyethoxy) acetate, rep III	0.49		1.27%

Table 3: Mean peptide depletion values for the positive control and the test chemical

Name, replicate number	Obtained mean % cysteine peptide depletion	Obtained mean % lysine peptide depletion	Mean % obtained peptide depletion
sodium (2-butoxyethoxy) acetate	3.56	1.32	2.44
CINNAMALDEHYDE	70.60	53.00	61.80

Interpretation of results:

other: no peptide depletion

Remarks:

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.

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to have no or minimal reactivity towards the synthetic peptides and thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

For the test chemical and positive control substance, in order to derive a prediction five independent tests were conducted, since the first run with cysteine peptide and the first two runs with lysine peptide did not meet acceptance criteria those results were rejected. All in all, the results of the two valid runs were used for the classification of the test item.

Peptide depletion resulted from the positive control cinnamaldehyde was 70.60 % with cysteine peptide and 53.00 % with the lysine peptide while the standard deviation (SD) of the percent peptide depletions were 1.05 % and 0.49 % for the cysteine and lysine peptides respectively in the valid runs. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine (0.50 – 0.49 mM) and lysine peptides (0.50 – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.6 % and 0.3 % percentages for the cysteine and lysine peptides. In the last runs for each peptide all validity criteria were also met, confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 3.56 % while the percent lysine peptide depletion was 1.32 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 2.44 %, which did not exceed the 6.38 % threshold of the applicable prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 September 2018 - 4 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

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
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, so that the final negative (solvent) control concentration was 1% DMSO in exposure medium on the treated plates.

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 one of these 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 Method:
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. There was no cytotoxicity induced by the positive control at any of the concentrations.

Key result

Run / experiment:

other: 1st

Parameter:

other: maximal fold Luciferase induction

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2nd

Parameter:

other: maximal fold Luciferase induction

Value:

1

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, the laboratory demonstrated technical proficiency, 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 no cytotoxicity induced by the test item at any of the concentrations.

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

Concentration (µM)	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)	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%

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

First test 18 September -21 September 2018

						Test item
Concentration (µM)	1	2	4	8	16	31	63	125	250	500	1000	2000
20180918-1117-2	1.95	1.06	1.11	1.00	1.13	1.12	0.88	0.96	1.07	0.82	1.08	1.11
Plate ID 20180918-1117-3	0.75	0.77	0.89	0.75	0.85	0.82	0.90	0.72	0.97	0.78	1.03	0.83
20180918-1117-4	0.91	0.91	0.79	0.92	1.19	0.85	0.73	0.92	1.00	0.85	0.65	0.79
average induction	1.20	0.91	0.93	0.89	1.06	0.93	0.84	0.86	1.01	0.82	0.92	0.91
significance	0.614	0.296	0.295	0.217	0.684	0.334	0.065	0.187	0.888	0.052	0.504	0.228
viability	109%	94%	96%	116%	97%	102%	96%	103%	94%	101%	117%	97%
Second test 01 October - 04 October 2018
						Test item
Concentration (µM)	1	2	4	8	16	31	63	125	250	500	1000	2000
20181001-1108-2	0.91	1.08	0.83	1.04	0.98	0.77	0.92	0.86	0.82	1.13	0.81	1.09
Plate ID 20181001-1108-3	0.77	0.71	0.96	0.97	0.72	0.79	0.80	0.69	0.70	0.91	0.86	0.87
20181001-1108-4	0.86	0.97	0.85	0.72	0.72	1.12	0.93	0.74	0.93	0.95	1.05	0.93
average induction	0.85	0.92	0.88	0.91	0.81	0.90	0.88	0.76	0.81	1.00	0.91	0.96
significance	0.084	0.465	0.306	0.611	0.165	0.248	0.114	0.044	0.031	0.972	0.219	0.725
viability	106%	103%	105%	106%	106%	104%	107%	110%	114%	106%	107%	119%

 

Interpretation of results:

other: no activation of keratinocytes

Remarks:

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.

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item sodium (2-butoxyethoxy) acetate 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 sodium (2-butoxyethoxy) acetate was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD TG 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 induced by the positive control at any of the concentrations.

For the test item twelve doses were used in two independent runs between 2000 μM to 1 μM. There was no cytotoxicity induced by the test item in KeratinoSens™ cells compared to the solvent/vehicle control. 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 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:

15 August - 30 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION – ANNEX I: IN VITRO SKIN SENSITISATION: 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:

Cell line used: THP-1 cells, Human Acute Monocytic Leukemia, LOT 63558119
Maintenance (culture) medium for THP-1 cells: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.05 mM L-glutamine solution and 0.05 mM 2-mercaptoethanol.
Material and conditions:
- For cell culture standard laboratory equipment was used.
- Flow cytometer: Apogee Flow Cytometer (APOGEE A60 UNIVERSAL ANALIZER)
- Buffer: Phosphate Buffered Saline (DPBS)
- Blocking Solution 0.01% Globulins Cohn fraction II,III
- Antibodies: FITC antibody solutions (50 μL of pre-mixed solution per sample):
- FITC labelled CD86 antibody solution - 6 μL of antibody to 44 μL of FACS buffer
- FITC labelled CD54 antibody solution - 3 μL of antibody to 47 μL of FACS buffer
- FITC labelled-mouse IgG1 antibody solution - 3 μL of antibody to 47 μL of FACS buffer
- Reagent for cytotoxicity test: Propidium iodide (PI)solution: 12.5 μg/mL of propidium iodide solution in DPBS

Controls used:
- Vehicle control: medium
- Positive control: 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7)

Preliminary tests:
For testing, THP-1 cells were seeded at a density of either 0.1 × 10E6 cells/mL or 0.2 × 10E6 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintance medium at 2 × 10E6 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 μL / wells (1 × 10E6 cells/well). The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37°C under 5% CO2. The plates were sealed with breathable microplate covers prior to the incubation to avoid evaporation of test item. After 24±0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample. The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. 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. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

Main Test (CD86 and CD54 expression):
Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 10E6 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours. 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 (250 g, 5 min, 4 ºC). 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 (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated 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.

Acceptance criteria:
- 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%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90% (when HSC is used instead of CV75, the data for the test chemical is accepted regardless the cell viability at the highest dose).
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.

Prediction model:
If the RFI of CD86 is equal to or greater than 150% at any tested dose (>50% of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200% at any tested dose (>50% of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise it is considered as negative.
In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.

Positive control results:

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

Key result

Run / experiment:

other: 1st run

Parameter:

other: RFI value for CD54 [%]

Value:

163

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 5000 µg/mL

Key result

Run / experiment:

other: 1st run

Parameter:

other: RFI value for CD86 [%]

Value:

97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 3472 µg/mL

Key result

Run / experiment:

other: 2nd run

Parameter:

other: RFI value for CD54 [%]

Value:

112

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 4166 µg/mL

Key result

Run / experiment:

other: 2nd run

Parameter:

other: RFI value for CD86 [%]

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 1395 µg/mL

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

Preliminary tests (Dose finding assays): Cell viability was not lower than 75% at any tested concentrations in either of the runs. Since not even the highest allowed concentration caused significant reduction in cell viability, no CV75 value could be determined. Therefore, the highest allowed stock concentration (500 mg/mL) was used for setting the dose-range for measuring CD86 and CD54 expression in the main test.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in Annex I of the Test Guideline 442E, 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 ranges. Moreover, a historical database of reactivity check results positive controls 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
- Acceptance criteria met for variability between replicate measurements: yes

Table 1:  Positive and negative control results

sodium (2- butoxyethoxy) acetate	sample	concentration	RFI		viability (IgGl)
			CD86	CD54	IgG
Exposure date: 28 August 2013	DMSO	0.2%	143	69	93.0
	DNCB	4.0 µg/mL	730	977	70.9
Exposure date: 30 August 2018	DMSO	0.2%	110	74	92.7
	DNCB	4.0 µg/mL	709	608	65.0

 

Table 2: Results 1st run

sodium (2-butoxyethoxy) acetate				Cells batch weighted in (g)		THP-1 (ATCC) MC2 20180205-20180803 0,5				Exposure date Analysis date		2018 August 27 2018 August 28
sample	Conc. (µg/mL)	MFI (Geo Mean)			corrected MFI		RFI (CD86)		RFI (CD54)			viability
							Top control		Top control
		CD86	CD54	isotype	CD86	CD54	vs medium control	vs DMSO control	vs medium control		vs DMSO control	IqG	CD86	CD54
control DMSO	0,2%	3153 3430	2786 2564	2321 2244	832 1186	465 320	100 143		100 69			90,4 93,0	91,5 92.2	95,3 94,9
DNCB	4,0	11456	5927	2802	8654	3125		730			977	70,9	71,2	72,8
sodium (2-butoxyethoxy) acetate	5000	3302	3396	2637	665	759	80		163			91,9	89,8	89,7
	4167	3245	3068	2582	663	486	80		105			89,1	90,1	91,9
	3472	3367	2990	2559	808	431	97		93			89,3	89,9	90,3
	2894	3229	2905	2501	728	404	88		87			91,4	92,5	92,9
	2411	3231	2896	2437	794	459	95		99			88,5	92,6	92,4
	2009	3202	2803	2520	682	283	82		61			91,3	91,2	93,0
	1674	3219	2892	2542	677	350	81		75			91,1	93,3	93,4
	1395	3210	2855	2464	746	391	90		84			91,5	93,3	93,9

Table 2: Results 2nd run

sodium (2-butoxyethoxy) acetate				Cells batch weighted in (g)		THP-1 (ATCC) MC2 20180205-20180803 0,4999						Exposure date Analysis date			2018 August 29 2018 August 30
		MFI (Geo Mean)								RFI (CD86)			RFI (CD54)
						corrected MFI			Top control				Top control			viability
	Conc. (µg/mL)								vs medium				vs medium
sample		CD86	CD54		isotype	CD86	CD54		control		vs DMSO control		control	vs DMSO control		IqG	CD86	CD54
control	-	3524	3521		2462	1062	1059		100				100			90,3	93,7	96,1
DMSO	0,20%	3509	3117		2338	1171	779		110				74			92,7	95,1	95,0
DNCB	4,0	10933	7374		2634	8299	4740				709			608		65,0	68,0	67,0
sodium (2-butoxyethoxy) acetate	4999	3719	3757		2731	988	1026		93				97			90,1	91,1	91,3
	4166	3581	3806		2622	959	1184		90				112			91,9	93,3	93,6
	3472	3532	3745		2618	914	1127		86				106			90,7	93,8	94,0
	2893	3416	3822		2786	630	1036		59				98			91,2	91,1	93,6
	2411	3505	3597		2596	909	1001		86				95			90,0	91,1	94,2
	2009	3424	3271		2509	915	762		86				72			91,7	92,9	94,0
	1674	3461	3271		2500	961	771		90				73			92,7	92,0	94,5
	1395	3576	3304		2469	1107	835		104				79			86,9	89,0	93,1

Interpretation of results:

other: no activation of dendritic cells.

Remarks:

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.

Conclusions:

Based on the results and the h-CLAT prediction model, the test item sodium (2-butoxyethoxy) acetate was negative (having a non-sensitizing potential) therefore considered not having potential to activate dendritic cells under the experimental conditions of the human Cell Line Activation Test.

Executive summary:

The skin sensitization potential of the test item sodium (2-butoxyethoxy) acetate was studied using the h-CLAT method according OECD TG 442E.

The extent of cytotoxicity induced by the test item in THP-1 cells was determined in two dose finding tests. CV75 could not be determined, since there was no test item concentration that resulted in 75% cell viability compared to the solvent/vehicle control. Thus the highest soluble concentration (500 mg/mL) was determined and used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in two independent runs between 5000 μg/mL - 1395 μg/mL.

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

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

For the test item the increase in CD86 marker expression (RFI) was lower than 150 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Therefore, both runs were negative for CD86 marker expression.

Also, the increase in CD54 marker expression (RFI) was lower than 200 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Based on the concordant negative results of the two runs, CD54 marker expression was not induced by the test item. Since none of the markers gave positive result, the overall h-CLAT prediction was concluded negative.

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. 

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

For the test chemical and positive control substance, in order to derive a prediction five independent tests were conducted, since the first run with cysteine peptide and the first two runs with lysine peptide did not meet acceptance criteria those results were rejected. All in all, the results of the two valid runs were used for the classification of the test item.

Peptide depletion resulted from the positive control cinnamaldehyde was 70.60 % with cysteine peptide and 53.00 % with the lysine peptide while the standard deviation (SD) of the percent peptide depletions were 1.05 % and 0.49 % for the cysteine and lysine peptides respectively in the valid runs. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine (0.50 – 0.49 mM) and lysine peptides (0.50 – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.6 % and 0.3 % percentages for the cysteine and lysine peptides. In the last runs for each peptide all validity criteria were also met, confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 3.56 % while the percent lysine peptide depletion was 1.32 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 2.44 %, which did not exceed the 6.38 % threshold of the applicable prediction model.

The skin sensitization potential of the test item sodium (2-butoxyethoxy) acetate was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD TG 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 induced by the positive control at any of the concentrations.

For the test item twelve doses were used in two independent runs between 2000 μM to 1 μM. There was no cytotoxicity induced by the test item in KeratinoSens™ cells compared to the solvent/vehicle control. 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 negative for luciferase gene induction.

The skin sensitization potential of the test item sodium (2-butoxyethoxy) acetate was studied using the h-CLAT method according OECD TG 442E.

The extent of cytotoxicity induced by the test item in THP-1 cells was determined in two dose finding tests. CV75 could not be determined, since there was no test item concentration that resulted in 75% cell viability compared to the solvent/vehicle control. Thus the highest soluble concentration (500 mg/mL) was determined and used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in two independent runs between 5000 μg/mL - 1395 μg/mL.The positive control gave positive results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50% in each run. The DMSO controls had negative outcomes compared to the medium control for both markers in all runs, meaning that the RFI values of both CD86 and CD54 expression was never over the positive criteria. For the test item the increase in CD86 marker expression (RFI) was lower than 150 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Therefore, both runs were negative for CD86 marker expression. Also, the increase in CD54 marker expression (RFI) was lower than 200 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Based on the concordant negative results of the two runs, CD54 marker expression was not induced by the test item. Since none of the markers gave positive result, the overall h-CLAT prediction was concluded negative.

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) and does neither activate keratinocytes (LuSens, OECD TG 422D) nor dendritic cells (h-CLAT, OECD TG 442E). Therefore, the substance is not predicted to be 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 as skin sensitising according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.