Tributylethylammonium ethyl sulphate

Endpoint summary

Description of key information

A DPRA and KeratinoSens^TM performed with TBEAES according to OECD guidelines and in accordance with GLP principles were negative. A DEREK assessment was negative. TBEAES DRY can therefore be concluded to have no skin sensitizing properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

QSAR

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

An in silico assessment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.

Principles of method if other than guideline:

A DEREK assessment was performed.

GLP compliance:

no

Justification for non-LLNA method:

Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined with a WoE. An in vivo test (LLNA) is only allowed as last resort. DEREK results are adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.

Key result

Parameter:

other: Prediction on skin sensitisation

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

DEREK NEXUS version 6.0.0 did not yield any alerts for skin sensitisation for the anion (ethyl sulfate). The anion does not contain any unclassified or misclassified features. The
cation (tributylethylammonium) yielded an alert for skin sensitisation based on the presence of the quaternary ammonium group (equivocal). It is important to note that the cation should be suitably lipophilic to exhibit skin sensitisation as a stable association with membrane lipids in forming an immunogenic complex is needed. The lipophilicity of the tributylethylammonium is expected to be too low and the alkyl chains too short to form a stable complex with membrane lipids. Moreover, the only analogue DEREK NEXUS has found has only 15% similarity with tributylethylammonium cation. Therefore, TBEAES DRY is considered to be not sensitising to the skin.

Interpretation of results:

study cannot be used for classification

Remarks:

(study is part of a weight of evidence approach and is not used for classification on its own)

Conclusions:

Based on the DEREK NEXUS assessment TBEAES is considered not sensitising to the skin.

Executive summary:

Based on the DEREK NEXUS assessment TBEAES is considered not sensitising to the skin.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 October 2017 - 24 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.

Details on the study design:

TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ) and MQ/ACN (1:1, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 53.06 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1563 µL MQ to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in an incubator at 25±2.5°C. After incubation, the samples were transferred to the autosampler. The incubation time between placement of the samples in the incubator and analysis of the first cysteine and lysine sample was 24.5 hours and 24 hours, respectively. The time between the first and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1. (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2. (See 'other information on materials and methods').

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 3), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: Mean SPCC depletion(%)

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.6%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 78.7% ±1.1%

Remarks on result:

other: SD: 6%

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean SPCL depletion (%)

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 2%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 50 ± 2.2%

Remarks on result:

other: SD: 0.8%

Other effects / acceptance of results:

No precipitation or co-elution occured during the tests.
See Table 4 & 5 "any other information on results incl. tables" for acceptibility criteria.

Table 4: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.996	>0.99	0.998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.501 ± 0.009	0.50 ± 0.05	0.488 ± 0.002
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.502 ± 0.013	0.50 ± 0.05	0.482 ± 0.009
Mean peptide concentration RC-Cwatersamples (mM)	0.50 ± 0.05	0.497 ± 0.006	0.50 ± 0.05	0.477 ± 0.006
CV (%) for RC samples B and C	<15.0	1.7	<15.0	2.0
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	73.7	40.2-69.0	50.4
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	2.2
SD of peptide depletion for  the test item (%)	<14.9	0.6	<11.6	0.8

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 5: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
TBEAES DRY	0.8%	±0.6%	1.0%	±0.8%	0.9%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

study cannot be used for classification

Remarks:

(study is part of a weight of evidence approach and is not used for classification on its own)

Conclusions:

In a DPRA study performed according to OECD TG 442C and in accordance with GLP principles, TBEAES was considered negative and classified in the "No or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico study, performed according to OECD guideline 442C and in accordance with GLP principles, the reactivity of TBEAES DRY towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers.

Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model.

MQ water was found to be an appropriate solvent to dissolve the test substance, and was therefore used in this DPRA study. Cinnamic aldehyde was used as a positive control. No precipitation was observed.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples, the CV for RC samples, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA. Therefore, the study was considered to be valid. No co-elution of the test item with SPPC or SPCL was observed.

In the cysteine reactivity assay the test item showed 0.8% SPCC depletion while in the lysine reactivity assay the test item showed 1.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.9% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 December 2017 - 15 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSensTM assay is recommended in international guidelines (e.g. OECD) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

Specific details on test material used for the study:

pH (1% in water, indicative range): 5.7-5.6

Details on the study design:

TEST ITEM PREPARATION
In the main experiments the test item was dissolved in DMSO at 200 mM (clear colourless).
From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with DMEM-glutamax. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

Test item concentrations were used within 3 hours after preparation.

CONTROL ITEMS
Positive control: Ethylene dimethacrylate glycol (tested in triplicate)
*Batch SHBG0572V
*Purity 98.3%
*Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of a solvent control of 1% DSMO were tested
- Blank control: on each plate three bank wells were tested (no cells and no treatment)

TEST DESIGN

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+8 in experiment 1 and P+10 in experiment 2.

Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.

Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in a plate reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment: For the KeratinoSens™ cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with a plate reader.


DATA ANALYSIS: according to guideline.

Interpretation of results :
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 repetitions.
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 μg/mL should be considered as inconclusive.

Acceptance criteria:
1) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
2) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol 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.
3) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.55 and the EC1.5 75 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.45 and the EC1.5 85 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.67

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: 1768 μM

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.63

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: 1819 μM

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was between 5 and 125 µM (75 µM and 85 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.55-fold and 2.45-fold in experiment 1 and 2, respectively).
Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (6.5% and 10.9% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

No cytotoxicity (no IC30 and IC50 value) or precipitation was oberserved.

Interpretation of results:

study cannot be used for classification

Remarks:

(study is part of a weight of evidence approach and is not used for classification on its own)

Conclusions:

In a KeratinoSens™ assay performed according to OECD TG 442D and in accordance with GLP principles TBEAES DRY is classified as negative since no relevant induction was observed up to test concentrations of 2000 µM.

Executive summary:

A KeratinoSens^TM assay was performed with the TBEAES DRY according to OECD 442D and in accordance with GLP principles. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed. It is concluded that the test conditions were adequate and that the test system functioned properly in both experiments.  The test item showed no toxicity (no IC₃₀ and IC₅₀ value). In experiment 1 and 2, no biologically relevant induction of luciferase activity was observed. The maximum induction was with 1.67-fold and 1.63-fold > 1.5-fold in experiment 1 and 2, respectively. The EC_1.5was with 1768 µM and 1819 µM in experiment 1 and 2, respectively, ≥ 1000 µM and therefore not relevant for the skin sensitization endpoint. In conclusion, the test item is classified as negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 6.0.0 did not yield any alerts for skin sensitisation for the anion (ethyl sulfate). The anion does not contain any unclassified or misclassified features. The cation (tributylethylammonium) yielded an alert for skin sensitisation based on the presence of the quaternary ammonium group (equivocal). It is important to note that the cation should be suitably lipophilic to exhibit skin sensitisation as a stable association with membrane lipids in forming an immunogenic complex is needed. The lipophilicity of the tributylethylammonium cation is expected to be too low and the alkyl chains too short to form a stable complex with membrane lipids. Moreover, the only analogue DEREK NEXUS has found has only 15% similarity with tributylethylammonium cation. Therefore, TBEAES DRY is considered to be not sensitising to the skin.

A valid DPRA test was performed according to OECD 442C and in accordance with GLP. For the DPRA assay TBEAES DRY was dissolved in Milli-Q water at 100 mM and formed a clear solution by visual inspection. No co-elution of the test item with SPCC or SPCL and no precipitation at start and end of incubation was observed. In the cysteine reactivity assay the test item showed 0.8% SPCC depletion while in the lysine reactivity assay the test item showed 1.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.9% and as a result the test item was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be negative in the DPRA.

A valid KeratinoSens^TM assay was performed according to OECD 442D and in accordance with GLP. For the KeratinoSens^TM assay TBEAES DRY was dissolved in DMSO to a final concentration of 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2 -fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed. The test item showed no toxicity (no IC₃₀and IC₅₀value). In experiment 1 and 2, no biologically relevant induction of luciferase activity was observed. The maximum induction was with 1.67-fold and 1.63-fold > 1.5-fold in experiment 1 and 2, respectively. The EC_1.5was with 1768 µM and 1819 µM in experiment 1 and 2, respectively, ≥ 1000 µM and therefore not relevant for the skin sensitization endpoint. TBEAES is classified as negative in the KeratinoSens^TMassay.

Based on the DEREK NEXUS assessment (not sensitizing), a negative DPRA assay and a negative KeratinoSens^TM assay, TBEAES DRY can be concluded to have no skin sensitizing properties. The U-SENS^TM assay, addressing the activation of dendritic cells would not yield additional information irrespective of whether the result would be negative or positive.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available results, TBEAES is not classified and has no obligatory labelling requirement for sensitization by skin contact according to the Regulation (EC) No 1272/2008.