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Description of key information

Skin sensitization (OECD 442C), direct peptide binding assay: not sensitizing

Skin sensitization (OECD 442D), activation of keratinocytes: not sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2017 - 3 Nov 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:
04 February 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
The test substance was dissolved in deionized water. Three samples of the test substance were incubated with each peptide (i.e., cysteine peptide or lysine peptide). Additionally, triplicates of the concurrent vehicle control were incubated with the peptides. These vehicle controls were prepared as three separates sets, set A, set B, and set C. Set A was analyzed together with the calibration samples without incubation and served as a performance control. Sets B and C were prepared and
incubated with the samples. Set B was placed at the very start and ending of the sample list and served as stability control of the peptide over the analysis time. Set C was analyzed with the samples and served for calculation of the peptide depletion of any chemical formulated in the vehicle.

Positive and co-elution controls also were evaluated. The positive control used was ethylene dim ethacrylate (50 mM) and the co-elution control was peptide buffer with the test substance but without peptide. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm.

For the assay to be considered valid, the following criteria needed to be met:
The standard calibration curve was an r² > 0.99.
The negative control (vehicle control) samples of sets A and C were 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C were < 15%.
The variability between the mean of three single samples was low (SD < 14.9% for % cysteine de
pletion and < 11.6% for % lysine depletion).
The positive control caused depletion of both peptides comparable to historic data.

For results to be considered negative, the mean peptide depletion is ≤ 6.38%. All other results are considered positive with low (> 6.38 ≤ 22.62%), moderate (> 22.62 ≤ 42.47%), or high (> 42.47%) reactivity. In the case where mean peptide depletion could be determined, but valid C-peptide depletion was available, a C peptide depletion of ≤ 13.89% was considered negative. All other results are considered positive with low (> 13.89 ≤ 23.09), moderate (> 23.09 ≤ 98.24), or high (> 98.24) reactivity.
Positive control results:
The positive control caused depletion of both peptides comparable to historic data.
Parameter:
other: mean cysteine peptide depletion
Value:
5.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean lysine peptide depletion
Value:
-0.53
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean of both peptide depletions
Value:
2.51
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test substance was dissolved in de-ionized water at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The mean peptide concentration of the three samples of set A was 0.505 ± 0.005 mM, demonstrating good performance. The mean peptide concentration of the three samples of set B, analyzed at the beginning of the sample list was calculated to be 0.485 ± 0.008 mM. The other three samples of set B, analyzed at the end of the sample list had a mean peptide concentration of 0.471 ± 0.014 mM. The CV of the 9 vehicle control samples of sets B and C was calculated to be 2.2%. Thus the peptide was considered stable over the time of analysis.

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
Interpretation of results:
other: no skin sensitising potential based on the key event “protein reactivity”
Conclusions:
Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model, it was concluded that Sodium 2-butoxyethyl sulphate shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2017 - 3 Nov 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:
February 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of study:
activation of keratinocytes
Details on the study design:
In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. No cytotoxicity was observed.
Based on these results, a main luciferase activity test was conducted in which activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. For each experiment, three replicates of each test-substance concentration were tested. After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were then placed into the incubator for the exposure period of 48 hours. For the luciferase assay, a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability. After the 48 hour exposure period, each test-substance concentration was visually inspected directly to detect test-substance precipitates. In additional to evaluating the test material, negative (DL-Lactic acid), vehicle (DMSO) and positive (ethylene glycol dimethacrylate) controls were evaluted concurrently.

For the luciferase assay, the supernatant was aspirated from the white assay plate, discarded, and the cells were washed twice after visual inspection of the cells. Subsequently, One-Glo-preparation was added and cells were shaken. After the incubation the luminescence was measured in the luminometer. For the statistical evaluation of luciferase fold induction the EXCEL-function T.TEST is used.

A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeded a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments. A test substance is considered to be negative when the criteria mentioned above were not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or maximum applicable concentration or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).

To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

For the cell viability MTT assay, cell culture medium was aspirated from all wells. The cells were then washed twice and thiazolyl blue tetrazolium bromide (MTT) solution was added to each well and further incubated for 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed. Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

For the assays to be considered valid, the following criteria needed to be met:
Tested concentration was not further evaluated when relative viability was less than 70%.
The cell viability of vehicle control cells was at least 85%.
The mean of the positive control EGDMA was ≥ 2.50 fold-induction and the mean of the LA < 1.50 and the mean of the viability was ≥ 70%.
The CV [%] of the luminescence in the vehicle control wells for each plate was below 20%.
The mean of the basal expression of the cells was < 1.50 fold-induction as compared to the solvent control.
In addition, positive, negative and vehicle control data were within the range of the historic data.
Positive control results:
The mean of the positive control EGDMA was ≥ 2.50 fold-induction.
Key result
Run / experiment:
other: 2nd experiment
Parameter:
other: ARE-dependent luciferase activity induction
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3rd experiment
Parameter:
other: ARE-dependent luciferase activity induction
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and min 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The 1st experiment was invalid for not meeting acceptance criteria in the PC and is not included in this report. the 2nd and 3rd experiments were considered valid.

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
Interpretation of results:
other: no skin sensitising potential based on the key event “inflammatory response in keratinocytes”
Conclusions:
In summary, after 48 hours of exposure to test substance Sodium 2-butoxyethyl sulphate luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance Sodium 2-butoxyethyl sulphate does not have a keratinocyte activating potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Two in vitro assays were conducted to assess the skin sensitization potential of Butylmonoglycol sulphate, Na-salt (CAS 67656-24-0). In a Direct Peptide Reactivity Assay (DPRA) conducted according to OECD 442C, the test substance was incubated with synthetic peptides for approximately 24 hours and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm (BASF, 2017a). For this assay, the test substance was dissolved in de-ionized water and 3 samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of vehicle control were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 5.02%. The mean K-peptide depletion, caused by the test substance was determined to be -0.53%. Based on these results, it was concluded that Sodium 2-butoxyethyl sulphate shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

In a LuSens assay, the keratinocyte activating potential of test substance Sodium 2-butoxyethyl sulphate was evaluated (BASF, 2017b). For this purpose the test substance was incubated with a luciferase reporter cell line for approximately 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. No cytotoxicity was observed.

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed and no precipitates were noticed in any concentration after 48 hours. In the two valid experiments, no biologically relevant ARE-dependent luciferase activity induction was observed. Therefore, it was concluded Sodium 2-butoxyethyl sulphate does not have a keratinocyte activating potential.

For the evaluation of skin sensitization potential three in vitro methods, addressing key events of the adverse outcome pathway for skin sensitisation as defined by the OECD have to be performed: protein reactivity (DPRA), activation of keratinocytes (LuSens), and activation of dendritic cells (h-CLAT). However, in the current case for sodium 2-butoxyethyl suphate the results derived with DPRA and LuSens were  sufficient for a final assessment. Therefore, further testing in h-CLAT was waived. Based on the negative results of the DPRA and LuSens sodium 2 -butoxyethyl sulphate is predicted not to be a skin sensitizer.

Justification for classification or non-classification

The available data on skin sensitization of Butylmonoglycol sulphate, Na-salt do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.