Potassium 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither bind to proteins in the DPRA nor activate the Nrf2 -Keap1 -ARE toxicity pathway (key event 2 of skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

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:

key study

Study period:

29 January 2019 - 04 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In chemico study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Name: Potassium 3,5,5-trimethylhexanoate
CAS No.: 93918-10-6
Batch No.: PURS151015
Purity: 99.4%
Physical State: solid
Colour: white
Molecular Weight: 196.33 g/mol
Storage Conditions: room temperature
Re-certification Date: 14 October 2019

Details on the study design:

This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC).
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical. This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.

Positive control results:

Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Key result

Run / experiment:

other: Test item / run 1

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Test item / run 2

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Test item / run 3

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

0.12

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Positive control / run 1

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

61.01

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Positive control / run 2

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

61.28

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Positive control / run 3

Parameter:

other: Depletion of the Lysine Peptide [%]

Value:

57.61

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Test item / run 1

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

1.16

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Test item / run 2

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Test item / run 3

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

1.06

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Positive control / run 1

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

70.09

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Positive control / run 2

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

70.27

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Positive control / run 3

Parameter:

other: Depletion of the Cysteine Peptide [%]

Value:

69.45

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Conclusions:

In this OECD 442 C DPRA study under the given conditions the test item showed minimal reactivity towards both peptides. The test item might be considered as “non-sensitiser”.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

In the present study Potassium 3,5,5-trimethylhexanoate was dissolved in dist. water, based on the results of the pre-experiments. Based on a molecular weight of 196.33 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation (small droplets) was observed for the samples of the positive control including the co-elution control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.39%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.95%. The controls confirmed the validity of the study for both, the cysteine and lysine run.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither bind to proteins in the DPRA nor activate the Nrf2 -Keap1 -ARE toxicity pathway (key event 2 of skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2019 - 04 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Name: Potassium 3,5,5-trimethylhexanoate
CAS No.: 93918-10-6
Batch No.: PURS151015
Purity: 99.4%
Physical State: solid
Colour: white
Molecular Weight: 196.33 g/mol
Storage Conditions: room temperature
Re-certification Date: 14 October 2019

Details on the study design:

The KeratinoSens assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.

Preparation of the Test Item

All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001336139). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube.
Vortex mixing was used to aid solubilisation. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Cell line

The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 02 in experiment 1; P 04 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

Composition of Media

Maintenance Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 2007923) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0298G)
- 1% geneticin (final concentration: 500 μg/mL; Gibco Life Science, Cat. No. 10131-027, Lot No.: 1894716)

Assay Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 2007923) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0298G)

Test Item Exposure Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 2007923) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0298G)

Experimental Procedure

A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity

After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability

For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Prediction Model

A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:

- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 μM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 μM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Positive control results:

Controls

A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO (AppliChem; Lot No.: 0001336139) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001336139) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean of experiment 1 and 2

Parameter:

other: Imax

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: IC30

Remarks:

[µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 2

Parameter:

other: IC30

Remarks:

[µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 1 & 2

Parameter:

other: IC50

Remarks:

[µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Positive control 1

Parameter:

other: EC1.5 PC

Value:

15.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Positive control 2

Parameter:

other: EC1.5 PC

Value:

17.73

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The parent study fulfills all acceptance criteria of this method.

CV Solvent Control (< 20%): 5.1 % / 7.9 %

No. of positive control concentration steps with significant luciferase activity induction >1.5 (≥ 1): 3.0 / 2.0

EC1.5 PC (± 2 x SD of historical mean 7 < x < 34 µM): 15.84 / 17.73

Induction PC at 64 μM (2 .00 < x < 8.00): 4.41 / 3.48

Results

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study Potassium 3,5,5-trimethylhexanoate was dissolved in DMSO. Based on a molecular weight of 196.33 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non-sensitiser.

The controls confirmed the validity of the study.

Table 1: Results of the cytotoxicity measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	100	100	100	0.0
Positive control	4.00 8.00 16.00 32.00 64.00	88.1 97.1 94.3 95.0 94.4	95.7 93.9 95.5 85.0 73.7	91.9 95.5 94.9 90.0 84.0	5.4 2.3 0.8 7.1 14.7
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	103.3 101.2 94.8 99.1 94.9 95.2 92.5 92.9 86.6 88.1 76.5 71.6	90.9 102.2 101.8 107.6 105.1 112.6 152.1 118.7 118.3 114.5 99.2 92.8	97.1 101.7 98.3 103.4 100.0 103.9 122.3 105.8 102.5 101.3 87.9 82.2	8.8 0.7 5.0 6.0 7.2 12.3 42.1 18.3 22.4 18.7 16.1 15.0

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [μM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.21 1.35 1.46 2.50 4.60	1.26 1.38 1.62 1.95 4.36	1.13 1.20 1.43 1.93 4.66	1.20 1.31 1.50 2.13 4.41	0.06 0.10 0.10 0.32 0.23	* * *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	1.10 0.91 0.92 1.01 1.05 1.05 1.02 0.97 0.96 0.94 0.74 0.65	1.22 1.09 1.20 1.11 1.08 1.12 1.11 1.01 1.15 0.90 0.83 0.67	1.12 1.03 1.06 0.96 1.05 1.05 0.91 0.95 0.94 0.90 0.80 0.67	1.15 1.01 1.06 1.03 1.06 1.07 1.01 0.98 1.02 0.91 0.79 0.66	0.06 0.09 0.14 0.08 0.02 0.04 0.10 0.03 0.12 0.02 0.05 0.01

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [μM]	Fold Induction					Significance
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.13 1.29 1.42 1.32 3.01	1.19 1.57 1.56 1.97 3.32	1.22 1.40 1.40 2.12 4.01	1.18 1.42 1.46 1.81 3.48	0.05 0.14 0.09 0.43 0.51	* *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.99 0.94 0.94 0.93 1.05 1.19 1.25 1.40 1.22 1.36 1.34 0.97	1.26 1.14 1.15 1.21 1.29 1.30 1.48 1.34 1.48 1.41 1.36 1.15	1.13 1.09 1.08 1.30 1.22 1.30 1.31 1.26 1.25 1.42 1.48 0.97	1.13 1.06 1.06 1.14 1.19 1.26 1.35 1.33 1.32 1.40 1.39 1.03	0.13 0.10 0.11 0.19 0.12 0.06 0.12 0.07 0.15 0.03 0.08 0.10

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.20 1.31 1.50 2.13 4.41	1.18 1.42 1.46 1.81 3.48	1.19 1.37 1.48 1.97 3.94	0.01 0.08 0.03 0.23 0.66	* *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	1.15 1.01 1.06 1.03 1.06 1.07 1.01 0.98 1.02 0.91 0.79 0.66	1.13 1.06 1.06 1.14 1.19 1.26 1.35 1.33 1.32 1.40 1.39 1.03	1.14 1.03 1.06 1.08 1.12 1.17 1.18 1.15 1.17 1.16 1.09 0.85	0.01 0.03 0.00 0.08 0.09 0.14 0.23 0.25 0.21 0.34 0.43 0.26

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In this KeratinoSens study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither bind to proteins in the DPRA nor activate the Nrf2 -Keap1 -ARE toxicity pathway (key event 2 of skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available