Sulfuric acid, C16-C18 (even numbered) alkyl esters, sodium salts and C16-18 (even numbered) alcohols

Endpoint summary

Description of key information

The registered substance was negative for sensitising properties in chemico in a DPRA assay and in vitro in an h-CLAT study in the human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure. An in vitro Keratinosens study with the registered substance in the ARE-Nrf2 luciferase test was tested positive based on the I-max of 1.80, however, the results are doubtful as Log P (>5) and cytotoxicity might have influenced the validity of the results, and there are supporting negative data for sensitisation potential from long chain alcohols (LCHO) and alkyl sulfates (AS) components, as described below. The Keratinosens study was therefore disregarded, whereas the DPRA and h-CLAT studies were used as weight-of-evidence for absence of sensitsation potential in combination with supporting information from components.

Supporting literature data for the long chain alcohols showed negative results for LCOH C14, C16 and C18. For the alkyl sulfates, C12-Alkylsulfate was positive in 2 LLNA studies tested up to 25% concentration, but not in a third LLNA study up to 25% concentration. The observed reactions were due to a non-antigen-specific proliferative stimulus induced by the irritating effect of the tested concentrations Sodium Lauryl sulfate can cause Langerhans’ cell migration from the epidermis to draining lymph nodes with a consequent transient increase in cell proliferation. Subsequent cell typing studies have shown the lymph node cell changes to be characteristic of irritancy and not of allergy. It is to be noted that the C12-Alkylsulfate is not part of the registered substance, but only used as supportive information that equivocal results were also observed, finally concluded to be non-sensitizing. For the longer AS C16-18, a guinea pig maximisation test was negative. Finally, there was no study for sodium sulfate, however this substance is endogeneous in the body.

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:

2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted February 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.59: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)

Version / remarks:

Commission Regulation (EU) No 2017/735 adopted February 14, 2017, published in the Official Journal of the European Union L 112/1, dated April 28, 2017.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: DPRA

Justification for non-LLNA method:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: OE70403001
- Expiration date of the lot/batch: April 2020
- Production date: April 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
PROCEDURE
1.Preparation of the cysteine or lysine-containing peptides
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.
2.Preparation of the test item
Solubility of the test item in an appropriate solvent was assessed before performing the assay. The test item was not soluble in acetonitrile or water. 78.16 mg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 were dissolved in 3 mL 2-propanol immediately before testing to prepare a 100 mM solution. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.
3.Positive control, reference controls and co-elution control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding 2-propanol to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.
4.Incubation of the test item with the cysteine and lysine peptide solutions
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis.
If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.
5.Preparation of the HPLC standard calibration curve
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
6.HPLC preparation and analysis
If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would have been noted as percent peptide depletion which would have been over-estimated leading to false positive predictions and/or assignment to a higher reactivity class. No dimerisation of the cysteine peptide occurred.
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.

Run / experiment:

other: test item solution incubated at 1:10 cysteine peptides

Parameter:

other: cysteine peptide depletion

Remarks:

expressed in %

Value:

4.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

considered negative according to the cysteine 1:10 prediction model

Run / experiment:

other: test item solution incubated at 1:50 lysine peptides

Parameter:

other: lysine peptide depletion

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No quantification of lysine peptide was possible as the test item-lysine peptide-reaction mixture formed a gel immediately after mixing the test item and peptide solution. For this reason, a homogeneous mixture of both solution components is very unlikely and, hence, the peptide concentration in the liquid parts of the above mentioned gel tends to contain a higher peptide concentration compared to the reference control C. This finding leads to the rating “interference”.

Other effects / acceptance of results:

Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde 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.
c) 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 or 2-propanol should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

The test item was dissolved at a concentration of 100 mM in 2-propanol.

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and 2-propanol were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding 2-propanol (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18-treated samples revealed a cysteine peptide depletion of 4.78%. No quantification of lysine peptide was possible as the test item-lysine peptide-reaction mixture formed a gel immediately after mixing the test item and peptide solution. For this reason, a homogeneous mixture of both solution components is very unlikely and, hence, the peptide concentration in the liquid parts of the above mentioned gel tends to contain a higher peptide concentration compared to the reference control C. This finding leads to the rating “interference”. Hence, the assessment was based on the cysteine 1:10 prediction model only.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 is considered negative and predicted to be a non sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. To verify that the solvent used to dissolve the positive control does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.50% for cysteine and 56.05% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

The linearity of the standard calibration curve was r2 = 0.9999 for cysteine peptide and for lysine peptide. Hence the requirement of r2 > 0.99 was met.

The mean peptide concentrations of reference control A were 0.509 or 0.492 mM cysteine or lysine peptide, respectively and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.

All acceptance criteria of validity were fulfilled in this test.

Interpretation of results:

GHS criteria not met

Conclusions:

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 revealed a mean cysteine peptide depletion of 4.78%. This value was below 13.89% - the threshold level for no or minimal reactivity - and hence, considered negative according to the cysteine 1:10 prediction model. No quantification of lysine peptide was possible because of interference with the test item. The test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The test item was dissolved at a concentration of 100 mM in 2-propanol.

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and 2-propanol were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding 2-propanol (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18-treated samples revealed a cysteine peptide depletion of 4.78%. No quantification of lysine peptide was possible as the test item-lysine peptide-reaction mixture formed a gel immediately after mixing the test item and peptide solution. For this reason, a homogeneous mixture of both solution components is very unlikely and, hence, the peptide concentration in the liquid parts of the above mentioned gel tends to contain a higher peptide concentration compared to the reference control C. This finding leads to the rating “interference”. Hence, the assessment was based on the cysteine 1:10 prediction model only.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 is considered negative and predicted to be a non sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. To verify that the solvent used to dissolve the positive control does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.50% for cysteine and 56.05% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

Conclusion

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 revealed a mean cysteine peptide depletion of 4.78%. This value was below 13.89% - the threshold level for no or minimal reactivity - and hence, considered negative according to the cysteine 1:10 prediction model. No quantification of lysine peptide was possible because of interference with the test item. The test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E

Version / remarks:

HUMAN CELL LINE ACTIVATION TEST (H-CLAT)

GLP compliance:

yes (incl. QA statement)

Type of study:

other: HUMAN CELL LINE ACTIVATION TEST (H-CLAT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: OE70403001
- Expiration date of the lot/batch: April 2020
- Purity test date: 21 november 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C


OTHER SPECIFICS:

Details on the study design:

1.Procedure
Preparation of cells
The human monocytic leukaemia cell line, THP-1 (TIB-202™, ATCC) was used for performing the h-CLAT assay. The cells were propagated and a working stock was prepared and stored in RPMI-1640 medium at -196°C containing 10% fetal bovine serum (FBS) , 0.1% 2 mercaptoethanol , 100 units/mL penicillin, 100 µg/mL streptomycin2 and 10% dimethyl sulfoxide (DMSO).
THP-1 cells were incubated at 37°C under 5% CO2 and humidified atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.1% 2 mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. The absence of mycoplasma contamination was regularly checked and confirmed. THP-1 cells were seeded 3 days prior to use at the density of 0.1 x 106 cells/mL. Prior to using them for testing, the cells were qualified by conducting a reactivity check. The reactivity check of the cells was performed using the positive controls (2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4)) and the negative control (lactic acid (LA)) at the earliest two weeks after thawing. Both DNCB and NiSO4 produce a positive response of both CD86 and CD54 cell surface markers; LA produces a negative response of both CD86 and CD54 cell surface markers. Only the cells which have pass the reactivity check were used for the assay. Cells can be propagated up to two months after thawing. Cells should not exceed 30 passages after thawing. The cells were used in passages between 67 and 71 after thawing (passage 49). The reactivity check was performed according to the procedures described in section 1.2.1.
For testing, THP-1 cells were seeded at a density of 0.1 x 106 cells/mL and pre-cultured in culture flasks for 72 hours. It is important that the cell density in the culture flask just after the pre-culture period is as consistent as possible in each experiment, because the cell density in the culture flask just after pre-culture could affect the CD86/CD54 expression induced by allergens. On the day of testing, cells harvested from the culture flask were resuspended with fresh culture medium at 2 x 106 cells/mL. Then, cells were distributed into a 24-well flat-bottom plate with 500 µL per well (1 x 106 cells/well).
1.1- Dose finding assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value was used to determine the concentration of test items for the CD86/CD54 expression measurement.
1.1.1- Preparation of test and control items
The test item was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mg/mL, the highest feasible concentration. The test item was soluble only by warming up to 37°C for 30 minutes. The test and control items were prepared on the day of testing.
For the h-CLAT assay, the stock solution of the test item in DMSO was diluted as follows: Eight dilutions (eight concentrations) were prepared, by two-fold serial dilution with DMSO (at 37°C). These dilutions were then further diluted 250-fold into culture medium containing 1% serum (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to achieve a further two-fold dilution and a final range of concentrations in the plate of 3.125 - 400 µg/mL). The highest feasible final concentration in the medium was 400 µg/mL. Test item precipitation was noted macroscopically at a concentration of 400 µg/mL medium.
The solvent was tested at a single final concentration in the plate of 0.2%. The final concentration of the vehicle in the culture system did not affect cell viability or growth rate. The control had undergone the same dilution steps as described for the working solutions which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.
1.1.2- Application of the test and control items
The culture medium or working solutions described in section 1.2.1 were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well flat-bottom plate. The treated plates were incubated for 24 ± 0.5 hours at 37°C under 5% CO2.
1.1.3- Propidium iodide (PI) staining
After 24 ± 0.5 hours of exposure 250 µL of each cell-preparation was transferred into a 96-well round-bottom plate and cells were collected by centrifugation (at 250 g for 5 minutes at 4°C). The supernatants were discarded and the remaining cells were washed twice with washing buffer (0.1% BSA in DPBS ). Cell pellets were resuspended in 400 µL washing buffer. Shortly prior to analysis 20 µL propidium iodide solution (12.5 µg/mL) were added (final concentration of PI: 0.6 µg/mL).
1.2 - Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The propidium iodide (PI) uptake was analysed using flow cytometry with the acquisition channel FL-3 (620 nm). A total of 10 000 living cells (PI negative) were acquired. The cell viability was calculated using the following equation by the cytometer analysis program. When the cell viability is low, up to 30 000 cells including dead cells were acquired. Alternatively, data can be acquired for one minute after the initiation of the analysis.
"Cell viability = " "Number of living cells" /"Total number of acquired cells" " x 100"
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
"Log CV75 = " "(75 - c) x log (b) - (75 - a) x log (d)" /"a - c"
where:
a is the minimum value of cell viability over 75%;
c is the maximum value of cell viability below 75%;
b and d are the concentrations showing the value of cell viability a and c respectively.
1.2.1- Preparation of the test and control items
The solvent/vehicle DMSO as determined in the dose finding assay was used to dissolve the test item. If the CV75 could not be determined (i.e. if sufficient cytotoxicity is not observed in the dose finding assay), the highest soluble or stably dispersed concentration of test item prepared with each solvent/vehicle would have been used as the starting concentration. The final concentration in the plate should not exceed 1000 µg/mL. The highest feasible final concentration in the medium was 400 µg/mL. The test item was first diluted to the concentration corresponding to 500-fold of the 1.2 × CV75 determined in the dose finding assay. Then, 1.2-fold serial dilution was made using the corresponding solvent/vehicle to obtain the dilutions (eight concentrations ranging from 500 x 1.2 x CV75 to 500 x 0.335 x CV75 were tested in the h-CLAT assay). The dilutions were then further diluted 250-fold into the culture medium (working solutions). These working solutions were further diluted two-fold for use for exposure in the plate. Only 24-well plates were used for CD86/CD54 expression measurement.
The solvent/vehicle control was prepared as described in section 1.2.1. DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. To obtain a 4.0 µg/mL concentration of DNCB in the plate, a 2 mg/mL stock solution of DNCB in DMSO was prepared and further diluted 250-fold with culture medium to a 8 µg/mL working solution.
1.2.2- Application of test and control items
For each test and control item, one experiment was needed to obtain a prediction. Each experiment consists of at least two independent runs for CD86/CD54 expression measurement. Each independent run was performed either on a different day or on the same day provided that for each run: a) independent fresh stock solutions and working solutions of the test item and antibody solutions were prepared and b) independently harvested cells were used (i.e. cells were collected from different culture flasks); however, cells may come from the same passage. Test and control items prepared as working solutions (500 µL) were mixed with 500 µL of suspended cells (1 x 106 cells) at 1:1 ratio, and cells were incubated for 24 ± 0.5 hours. In each run, a single replicate for each concentration of the test and control items were sufficient because a prediction was obtained from at least two independent runs.
1.2.3- Cell staining and analysis
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, collected by centrifugation (at 250 g for 5 minutes at 4°C), and then washed with washing buffer. After washing, cells were blocked with 600 µL of blocking solution (washing buffer containing 0.01% (w/v) globulin (Cohn fraction II, III, Human)) and incubated at 4°C for 15 minutes. After blocking, cells were divided into three aliquots of 180 µL into a 96-well round-bottom plate or microtube.
After centrifugation, cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies at 4°C for 30 minutes. The antibodies described in the h-CLAT DB-ALM protocol no. 158 were used (3:25 (v/v, for CD86 (BD-PharMingen, #555657; Clone: Fun-1)) or 3:50 (v/v, for CD54 (DAKO, #F7143; Clone: 6.5B5) and IgG1 (DAKO, #X0927)) with staining buffer). After washing with 200 µL of washing buffer twice, cells were resuspended in 400 µL washing buffer and stored until analysis at 4°C. Shortly prior to analysis, 13 µL of PI solution (final concentration of approximately 0.6 µg/mL) were added. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.
2- Evaluation
2.1- Data evaluation
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 positive control cells and test item-treated cells were calculated according to the following equation:

"RFI=" "MFI of test item-treated cells - MFI of test item-treated isotype control cells x100 " /"MFI of solvent/vehicle-treated cells - MFI of solvent/vehicle-treated isotype control cells"

The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) were also calculated according to the equation in section 1.3.

2.2 - Interpretation of results and prediction model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction (positive or negative). An
h-CLAT prediction was considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the
h-CLAT prediction was considered negative:
The RFI of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥ 50%);
The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥ 50%).
Based on the above conditions, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered positive and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered negative (with due consideration of the provisions in section 2.3 regarding negative results) without the need for a third run. The first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction was based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered positive.
For the test items predicted as positive with the h-CLAT, optionally, two Effective Concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which the test items induced a RFI of 150 or 200, may be determined. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA (Integrated Approach to Testing and Assessment). They can be calculated by the following equations:
"EC150 (for CD86) =" 〖" B" 〗_"concentration" " + [(150 -" 〖" B" 〗_"RFI" ") / (" "A" _"RFI " "- " "B" _"RFI" ") x (" "A" _"concentration" "-" "B" _"concentration" ") "
"EC200 (for CD54) =" 〖" B" 〗_"concentration" " + [(200 -" 〖" B" 〗_"RFI" ") / (" "A" _"RFI " "- " "B" _"RFI" ") x (" "A" _"concentration" "-" "B" _"concentration" ") "
where
A concentration is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
B concentration 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)
EC150 and EC200 can be calculated based on two independent runs (if just two runs were sufficient for the prediction of skin sensitation). For the purpose of more precisely deriving the EC150 and EC200 values, a third independent run for CD86/CD54 expression measurement can be performed if required by the Sponsor. The final EC150 and EC200 values are then determined as the median value of the ECs (in case of three runs) or the higher EC150 or EC200 of the two calculated values (in case of two runs).

2.3 - Acceptance criteria
The following acceptance criteria should be met when using the h-CLAT assay.
The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). RFI values of the solvent/vehicle control are calculated by using the formula described in section 6.1.
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of less than 90% at the highest concentration tested (i.e. 1.2 x CV75 according to the serial dilution scheme. If the cell viability at 1.2 x CV75 is equal to or above 90%, the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is above 90%.

Key result

Run / experiment:

other: h-CLAT

Parameter:

other: Average CV75

Remarks:

µg/mL

Value:

58 µg/mL

Vehicle controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Remarks:

DNCB

Other effects / acceptance of results:

The h-CLAT prediction was considered negative as:
•The RFI of CD86 was below 150% at any tested concentration (with cell viability ≥ 50%);
•The RFI of CD54 was below 200% at any tested concentration (with cell viability ≥ 50%).

Interpretation of results:

GHS criteria not met

Conclusions:

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 did not reveal any sensitising properties in the h-CLAT method.

Executive summary:

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18was examined for sensitising properties in theh-CLAT assay. The h-CLAT assay is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure to the test chemical.These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control was calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

The human monocytic leukaemia cell line, THP-1 (TIB-202™, ATCC) was used for performing the h-CLAT assay.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mg/mL. The test item was soluble only by warming up to 37°C for 30 minutes. The highest feasible final concentration in the medium was 400 µg/mL. Test item precipitation was noted macroscopically at a concentration of 400 µg/mL medium. A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.

Eight dilutions (eight concentrations) were prepared, by two-fold serial dilution with DMSO and a final range of concentrations in the plate of 3.125 - 400 µg/mL medium. DMSO was used as solvent control tested at a single final concentration in the plate of 0.2%.The CV75 value was used to determine the concentration of test item for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) an average CV75 of 58.0 µg/mL and a maximum test concentration for the main experiment of 69.6 µg/mL was calculated. Hence, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18was tested at 8 concentrations in the range from 19.4 to 69.6µg/mL. DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.

The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control.

The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was also calculated.

Acceptance criteria and evaluation of the h-CLAT assay parameters in the first and second experiment:

 

Parameter	RFI		MFI relative to IgG [%]		Viability Mean of IgG, CD54 and CD86 [%]
	CD54^#1	CD86^#1	CD54	CD86
Medium control	<150	<150	>105	>105	>90
Vehicle control	100	100	>105	>105	>90
Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18	<200	<150^#2	>105	>105	>50^#3
Positive control (DNCB)	>200	>150	>105	>105	>50

 

^#1         compared to the vehicle control

^#2           in both experiments at 69.6 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL a CD86 value of >150 (RFI = 221 or 266 and a mean viability of 28.1% or 40.6% (cytotoxic) was noted in the 1. or 2. experiment, respectively).       

^#3        >50% in at least 4 concentrations

The h-CLAT prediction was considered negative as:

·        The RFI of CD86 was below 150% at any tested concentration (with cell viability ≥ 50%);

·        The RFI of CD54 was below 200% at any tested concentration (with cell viability ≥ 50%).

The vehicle control and the positive control DNCB were run in both experiments. All quality criteria for the vehicle control and the positive control required were fulfilled.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 did not reveal any sensitising properties in the h-CLAT test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A key in chemico Direct Peptide Reactivity Assay (DPRA) to quantify cysteine- or lysine- peptide depleation following 24 ± 2 hours incubation was conducted with the registered substance (Rehders, 2019). The test item was dissolved at a concentration of 100 mM in 2-propanol.Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and 2-propanol were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding 2-propanol (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate. The test item revealed a cysteine peptide depletion of 4.78%. No quantification of lysine peptide was possible as the test item-lysine peptide-reaction mixture formed a gel immediately after mixing the test item and peptide solution. For this reason, a homogeneous mixture of both solution components is very unlikely and, hence, the peptide concentration in the liquid parts of the above mentioned gel tends to contain a higher peptide concentration compared to the reference control C. This finding leads to the rating “interference”. Hence, the assessment was based on the cysteine 1:10 prediction model only. In conclusion, a mean cysteine peptide depletion was 4.78%. This value was below 13.89% - the threshold level for no or minimal reactivity - and hence, considered negative according to the cysteine 1:10 prediction model. No quantification of lysine peptide was possible because of interference with the test item. The test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the DPRA.

A key in vitro h-CLAT study with the registered substance was done for sensitising properties in the human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure (Spruth, 2018a).These surface molecules are typical markers of monocytic THP-1 activation and may mimic Dendritic Cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control was calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers. The test item was dissolved in dimethyl sulfoxide (DMSO) with a highest feasible final concentration in the medium of 400 µg/mL. A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 was 58.0 µg/mL and a maximum test concentration for the main experiment of 69.6 µg/mL was calculated. Hence the test item was tested at 8 concentrations in the range from 19.4 to 69.6µg/mL. The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control. The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was also calculated. The results were as follows:

·        The RFI of CD86 was below 150% at any tested concentration (with cell viability ≥ 50%);

·        The RFI of CD54 was below 200% at any tested concentration (with cell viability ≥ 50%).

In conclusion, the registered substance did not reveal any sensitising properties in the h-CLAT test method. 

An in vitro Keratinosens study with the registered substance was done for sensitising properties in the ARE-Nrf2 luciferase test method (Spruth, 2018b). The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. The test item was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 40 mg/mL.

Two endpoints were measured: luciferase induction after a 48 hour treatment and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test item in DMSO as solvent. For Luciferase induction the maximal fold-induction over solvent control (I-max) and the concentration needed to reach an 1.5 fold induction (EC-1.5) were calculated. For cytotoxicity the IC-50 and IC-30 values were interpolated.

The following results were obtained:

-      Luciferase determinations: Imax = 1.80* ± 0.08; EC1.5 = 3.63 ± 0.63 µg/mL

-      Cytotoxicity determinations: IC50 = 15.30 ± 4.70 µg/mL; IC30 = 13.30 ± 3.90 µg/mL.

The KeratinoSens-TM prediction of the test item is considered positive based on the I-max of 1.80 which is higher than 1.5 fold and statistically significantly different (*) as compared to the vehicle control, the cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity above 1.5, the EC-1.5 value of 3.63µg/mL is less than 200 µg/mL and there is an apparent overall dose-response for luciferase induction.

However, the results are considered to be questionabls as Log P might influence the validity of the results and there are extensive supporting negative data for sensitisation potential from long chain alcohols (LCHO) and alkyl sulfates (AS), as described below. The results from the Keratinosens are contradictive to existing results on components, therefore the study is disregarded and classificiation is considered inconclusive.

Various literature data were available for the long chain alcohols (LCHO) with chain length between C14 and C18, as summarised below.

-      For LCOH 14 (Alfol 14; 1-Tetradecanol;CAS 112-72-1) no skin sensitisation reactions were observed in guinea pigs (SIDS, 2006; Iihama, 1997b).

-      For LCOH 16 (1-Hexadecanol; CAS 36653-82-4) no skin sensitisation reactions were observed in guinea pigs (SIDS, 2006; Driscoll 1996a;Gloxhuber,1983).

-      For LCOH 18 (1-Octadecanol; CAS 112-925) no skin sensitisation reactions were observed in guinea pigs (SIDS, 2006; Driscoll 1996a;Driscoll 1996b).

For the alkyl sulfates (AS), there were data for the C16-C18 chain lengths, and also for the C12 chain length, however the latter group was out of the scope of the registered substance. The C12 AS is considered as a worst case substance.

-      C12-Alkylsulfate (CAS 151-21-3) was tested in mice on the dorsum of the ears with 25µL of test material, in a first study (5,10 or 25%solution in DMSO) and in a second study (4 , 5, 10 or 25 % solution in DMF) (OECD SIDS 2007; Basketter et al.,1994; Montelius et al., 1994). Stimulation indices were 3.2, 4 and 4.2 in the first study and 4.0-4.1, 5.1-5.1 and 6.7-7.6 in the second study. Based on the stimulation index it was considered that C12-Alkylsulfate (SDS) was skin sensitizing, however the changes were considered to be characteristics of irritancy, not of allergy. In a third study C12-Alkylsulfate (CAS 151-21-3) was tested in mice on the dorsum of the ears with a) daily topical application of 25 ul of a 5, 10 or 25 % solution in water on 3 consecutive days or b) intradermal injection of 50 ul of a 0.05, 0.5 or 5 % solution in saline; after 5 days daily topical application of 25 ul of a 5 % solution in 50 % DMSO on 3 consecutive days. Stimulation indices were a) 0.73, 1.61, 1.13 and b) 1.58, 1.93, 1.48. Based on the stimulation indices it is considered that C12-Alkylsulfate (SDS) is not skin sensitizing (OECD SIDS 2007; Ikarashi et al., 1993). It is to be noted that -       C12-Alkylsulfate (CAS 151-21-3) is not part of the registered substance, but only used as supportive information that equivocal results were also observed, finally concluded to be non-sensitizing. The observed reactions were due to a non-antigen-specific proliferative stimulus induced by the irritating effect of the tested concentrations Sodium Lauryl sulfate can cause Langerhans’ cell migration from the epidermis to draining lymph nodes with a consequent transient increase in cell proliferation. Subsequent cell typing studies have shown the lymph node cell changes to be characteristic of irritancy and not of allergy (Basketter, 2002).

-      For LCOH 16-18 (Sulfuric acid, mono-C16-18-alkyl esters, sodium salts; CAS 68955-20-4), a guinea pig maximisation test was negative (OECD SIDS, 2007; Henkel 1986a & 1995).

For the sodium sulfate, no valid study was identified for skin sensitisation potential with sodium sulfate in animals (SIDS, 2005).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), classification of the registered substance is not warranted for skin sensitisation.