Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts

Endpoint summary

Description of key information

Skin sensitsation, in-vitro (KeratinoSens assay, 48 -hour exposure): Predicted non-sensitiser

Skin sensitisation, in-vitro (h_CLAT): Predicted sensitiser

Skin sensitisation, in-vivo (LLNA): Sensitister, EC3 = 9.3%

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 April 18 - 20 April 18

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Details on the study design:

Skin sensitisation (In vitro test system) - The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers.

Details on study design:
Test item - Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was determined by solubility testing during the study.
Reference items - Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
Exposure time: 48 ± 2h
Repetitions: Three - Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.

Test results are acceptable if:
• The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
• The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
o Average induction in the three replicates for cinnamic aldehyde at 32 µM is between the XCellR8 historical range (currently 1.6 and 3)
o EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded.
If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
•CV% of blank values < 20%

Positive control results:

All of the formal acceptance criteria of the tests were met except for acceptance criterion 2. However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.

Key result

Run / experiment:

other: Sorpol 5115 skin sensitisation

Parameter:

other:

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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:

GHS criteria not met

Conclusions:

In this study, the result of the skin sensitisation KeratinoSens test of SORPOL 5115 was negative.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 April 2018 - 24 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

Qualifier:

according to guideline

Guideline:

other: Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency.

Version / remarks:

Toxicol In Vitro. 2012 Oct; 26(7):1150-60

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Dose finding study
Test item concentrations for the main experiment (h-CLAT) were determined by two XTT tests. The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria.

The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100. After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.
At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader. The absorbance was measured at 450 nm (reference wavelength 690 nm).

Three independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values (second and third XTT test) was used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (μg/mL) were used for the test item in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

The test item was tested in three independent runs.

For the test item exposure the highest dose solution calculated from the second and third XTT assays was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Before using the flow cytometer, the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using software. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)

The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Positive control results:

The RFI values of the positive controls (Dinitrochlorobenzene, DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the third h-CLAT run exceeded the positive criteria.

Key result

Run / experiment:

other: h-CLAT Run 1

Parameter:

other: RFI of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative at all concentrations tested

Key result

Run / experiment:

other: h-CLAT Run 1

Parameter:

other: RFI of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative at all concentrations tested

Key result

Run / experiment:

other: h-CLAT Run 2

Parameter:

other: RFI of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative at all concentrations tested

Key result

Run / experiment:

other: h-CLAT Run 2

Parameter:

other: RFI of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Positive at 5.4, 7.8, 11.2, 16.2 and 19.4 µg/mL

Key result

Run / experiment:

other: h-CLAT Run 3

Parameter:

other: RFI of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative at all concentrations tested

Key result

Run / experiment:

other: h-CLAT Run 3

Parameter:

other: RFI of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Positive at 13.5 and 16.2 µg/mL

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization Adverse Outcome Pathway, AOP) of SORPOL 5115 dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of SORPOL 5115 was previously determined by two XTT tests.

In the first XTT test cytotoxic effects were observed in all tested test item concentrations (highest tested concentration 5000 μg/mL). Therefore the highest tested test item concentration was adjusted to 50 μg/mL. Cytotoxic effects were observed in the second and the third XTT test, starting with a test item concentration of 25 μg/mL up to the highest tested concentration (50 μg/mL; threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 16.2 μg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 5.4, 6.5, 7.8, 9.4, 11.2, 13.5, 16.2 and 19.4 μg/mL

The test item with an estimated log Pow (KOWWIN v1.68) of 1.64 (for C14 H27 O3 S1 Na1) and 0.32 for (C14 H29 O4 S1 Na1) was tested in 3 independent runs. The RFI of CD86 was equal or greater than 150% in at least one concentration of 2 out of 3 independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the third h-CLAT run exceeded the positive criteria.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Interpretation of results:

other: Positive for the third key event of the skin sensitisation Adverse Outcome Pathway

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 2018 - 29 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Version adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) 400/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: None indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): At least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
- IN-LIFE DATES: From: 15 August 2018 To: 29 August 2018

Vehicle:

other: 1% Pluronic in distilled water

Concentration:

5, 10, and 25% (w/w)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Solubility was assessed in acetone / olive oil (4:1), dimethyl formamide, butanone, dimethyl sulphoxide, acetone, 1% Pluronic L92 in distilled water, ethanol / water (7:3) and propylene glycol. 1% pluronic in water was found to be the only solvent which formed a solution suitable for dosing.
- Irritation: A 25% solution of the test material in the vehicle was found not to cause local skin irritation (observation for 6 days after start of exposure)
- Systemic toxicity: No indication of systemic toxicity
- Ear thickness measurements: Mean change in ear thickness of 23.81% during the 6 days over which measurements were taken. As the change was less than 25% this was concluded not to indicate irritation

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Key result

Parameter:

SI

Value:

1.38

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

3.27

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

4.97

Test group / Remarks:

25%

Key result

Parameter:

EC3

Value:

9.3

Cellular proliferation data / Observations:

5%, Stimulation Index = 1.38
10%, Stimulation Index = 3.27
25%, Stimulation Index = 4.97

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A skin sensitization study was conducted (XCellR8 Limited, 2018, Study 17EN20) to determine the potential for Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts to cause dermal effects. The KeratinoSens test method was performed according to OECD test guideline 442D and in compliance with GLP.

A single application of 12 concentrations of Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts were applied in cell culture medium with a final concentration of DMSO of 1%. Sensitization was determined in terms of keratinocyte activation. The luciferase measurements and MTT viability were measured at the end of the 48 hours exposure period.

The sensitisation potential of Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts was quantified by calculating EC1.5.

Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts did not induce luciferase >1.5 in any of the 3 repetitions, therefore the KeratinoSens result for Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts was negative.

Sulfonic acids, C14-18-alkane hydroxy and C12-20-alkapolyene and C14-18-alkene and C12-20-alkene hydroxy, sodium salts was not considered sensitizing.

A second in-vitro study to assess the potential of the substance to induce Skin Sensitisation was conducted according to the h-CLAT method (Envigo, 2018, Study Reference MP28DF). The study was conducted in accordance with OECD Test Guideline 442E and in compliance with GLP. Sorpol 5115 was assessed in three independent runs to measure the potential to induce the expression of CD54 and CD86, which may indicate the potential to induce skin sensitisation. The test item was found to have produced a positive result for CD86 in one or more concentrations in 2 out of the 3 runs. On this basis it was concluded that Sorpol 5115 was positive for the potential to induce skin sensitisation.

As a positive result was observed in one out of two available in-vitro skin sensitisation studies, an in-vivo (LLNA) study was conducted for confirmation purposes.

A murine Local Lymph Node Assay (LLNA) was conducted on Sorpol 5115 to confirm whether or not the substance has the potential to induce skin sensitisation. The LLNA was conducted according to OECD test guideline 429 and EC method B42, and was performed in compliance with GLP. Groups of four mice/level were treated with the test item at concentrations of 5, 10 or 25% in 1% pluronic in water. A further group of mice was treated with the vehicle alone.

The Stimulation Index was found to be less than 3 at 5%, and greater than 3 in the 10% and 25% groups. The EC3 value was determined to be 9.3%. On the basis that an SI greater than 3 was determined during the test it was concluded that Sorpol 5115 is positive for the potential to induce skin sensitisation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Sorpol 5115 was found to have induced skin sensitisation in a Local Lymph Node Assay in mice (EC3 = 9.3%). With reference to tables 3.4.3 and 3.4.4 or Annex I of the CLP Regulation (Regulation (EC) 1272/2008 including amendments to the 4th May 2017), as the EC3 is greater than 2% the substance meets the criteria for classification for Skin Sensitisation category 1B.