6-tert-butyl-1,1-dimethylindan-4-yl methyl ketone

Endpoint summary

Description of key information

Skin sensitization (LLNA, OECD TG 429): not sensitizing

Skin sensitization (DPRA, OECD TG 442C): negative, no indication for sensitisation

Skin sensitization (KeratinoSens, OECD TG 442D): positive, indication for sensitisation

Skin sensitization (h-CLAT, OECD TG 442E): inconclusive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

A LLNA study is conducted as the result of the in vitro/in chemico skin sensitisation testing battery was inconclusive and therefore not adequate for classification.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2017 to 12 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A LLNA study is conducted as the result of the in vitro/in chemico skin sensitisation testing battery was inconclusive and therefore not adequate for classification.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

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:

Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 oC and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test item at concentrations of 25%, 10% or 5% w/w in acetone/olive oil 4:1

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a maximum attainable concentration of 25% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included in "Any other information on materials and methods incl. tables". Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of five mice were treated with test item at concentrations of 25%, 10% or 5% w/w in acetone/olive oil 4:1. The vehicle acetone/olive oil 4:1 was chosen as it produced the highest concentration that was suitable for dosing. In a vehicle determination trial, the maximum attainable concentration was determined to be 25% in acetone/olive oil 4:1. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

A routine positive control study has been performed using the reduced LLNA. A group of five animals was treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 oC, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>=0.05 (not significant)

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of 5.66 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1. Based on the SI > 3, α-Hexylcinnamaldehyde, tech., 85% was considered to be a skin sensitizer under the conditions of the test.

Key result

Parameter:

SI

Value:

1.65

Remarks on result:

other: 5%

Key result

Parameter:

SI

Value:

1.55

Remarks on result:

other: 10 %

Key result

Parameter:

SI

Value:

2.37

Remarks on result:

other: 25 %

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

SI >= 3 was not reached

Cellular proliferation data / Observations:

Clinical Observations and Mortality Data:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight:
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.

Preliminary Screening Test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in acetone/olive oil 4:1.

Interpretation of results:

other: Not classified, criteria not met

Remarks:

according to EU CLP 1272/2008 and its amendments

Conclusions:

The test item, tested up to the maximum attainable concentration of 25% w/w in acetone/olive oil 4:1, was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay and GLP. Following a vehicle determination trial, the maximum attainable concentration was determined to be 25% in acetone/olive oil 4:1. The maximum attainable concentration was 25% because the substance is a solid and wetting/dilution is needed to apply the substance on the mouse ear. In a preliminary screening test no clinical signs of toxicity were noted at this maximum attainable concentration. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. At 5, 10 and 25% the substance showed SI values of 1.65, 1.55 and 2.37, respectively. The test item failed to produce a threefold or greater increase in 3HTdR incorporation compared to control values and is therefore considered to be a non-sensitiser under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 March 2017 - 10 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

15/03/2017

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Information from in vitro/in chemico test method(s) are preferred above the animal mouse LLNA study since 2016 according to Article 13(3) of regulation (EC) No 1907/2006. However, due to an inconclusive result from the in vitro/in chemico testing battery an LLNA has been performed as well.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: Ac-RFAAKAA-COOH, lot number 1556172 , purity 94% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).

Positive control: cinnamic aldehyde lot number MKBR2427V, purity > 95%.

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution. An additional control sample of 5 mM of the test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control so confirming that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution. An additional control sample of 25 mM of the test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control so confirming that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Incubation:
The appearance of the test substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis:
The concentration of both the cysteine and lysine peptides in the presence of the test substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 µm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes

Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples (x 100)) / (Mean peptide peak area of reference (stability) control samples)]

Positive control results:

71.8% depletion (SD = 0.43%, n = 3) and 58.4% depletion (SD = 0.45%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

Key result

Run / experiment:

other: Main experiment

Parameter:

other: cysteine depletion, %

Value:

-1.09

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Main experiment

Parameter:

other: lysine depletion, %

Value:

-1.58

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Solubility assessment:
The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed by visual inspection.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, criteria for reference (stability) controls and precision controls of both peptides were met (CV 0.87%, n = 6 and CV 0.45%, n = 6, for cysteine and lysine, respectively, at 0.50 mM).
- Acceptance criteria met for positive control: yes, 71.8% depletion (SD 0.43%, n = 3) and 58.4% depletion (SD 0.45%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.76% and 1.02%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:
Mean depletion of -1.09% and -1.58% was observed for the test substance with cysteine and lysine peptides, respectively. The mean result is therefore considered zero and with effectively no depletion of either peptide in the presence of the test item, the test item is placed in the reactivity class of “no to minimal reactivity” and thus is predicted to be negative by DPRA.
No co-elution peaks were observed in either the Cysteine or Lysine assays.

Interpretation of results:

other: DPRA was negative

Conclusions:

It can be concluded that this DPRA test is valid and with effectively no depletion of either Cysteine nor Lysine peptide in the presence of the test item, the test item was classified in the "no to minimal reactivity" class based on the DPRA prediction model and is thus negative in the DPRA.

Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance were successfully analysed by the validated DPRA analytical method. In the Cysteine reactivity assay, Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM test substance, 0.5 mM Cysteine. In the Lysine reactivity assay, Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in Lysine peptide stock solution. The final sample concentration was 25 mM test substance, 0.5 mM Lysine. The individual peptide depletion results for the test substance were -1.09% (Cysteine) and -1.58% (Lysine). The mean result is therefore considered zero. All analytical acceptance criteria of the test were met. No co-elution peaks were observed in either the Cysteine or Lysine assays. With effectively no depletion of either peptide in the presence of the test substance, the test substance was classified in the “no to minimal reactivity” class based on the DPRA prediction model and was thus considered to be negative in the DPRA.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 May, 2017 - 30 June, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Information from in vitro/in chemico test method(s) are preferred above the animal mouse LLNA study since 2016 according to Article 13(3) of regulation (EC) No 1907/2006. However, due to an inconclusive result from the in vitro/in chemico testing battery an LLNA has been performed as well.

Details on the study design:

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

Controls:
Positive control: Ethylene dimethacrylate glycol, 7.8-250 µM, tested in triplicate, final concentration DMSO of 1%.
Negative control: DMSO (vehicle) (1% in exposure medium).
Blank: On each plate three blank wells were tested (no cells and no treatment) to assess background values.

Number of replicates: three independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (the passage numbers used were 25 in experiment 1, 16 in experiment 2 and 24 in experiment 3) and are employed for routine testing using the appropriate maintenance medium. Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Test item preparation:
No correction was made for the composition/purity of the test item. A solubility test was performed. The test item formed a non-homogenous suspension in DMSO at a concentration of 200 mM. Therefore, this concentration was not suitable to test. The test item was dissolved in DMSO at a concentration of 100 mM. The 100-fold dilution in DMEM of 100 mM formed a homogeneous solution (slight precipitation). This concentration was selected as highest concentration for the first experiment assay.
In the first experiment, the test item was dissolved in DMSO to a final concentration of 100 mM. The compound formed a clear colourless solution at 100 mM. In the first experiment, final test concentrations of 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 and 0.49 μM (final concentration DMSO of 1%) were tested. All concentrations of the test item were tested in triplicate. Precipitation of the test item was observed at the dose levels of 250, 500 and 1000 μM at the start of the treatment. No precipitation was observed at the end of the treatment.
In the second and third experiment, the test item was dissolved in DMSO at 12.5 mM. Final test concentrations were 125, 100, 80, 64, 51, 41, 33, 26, 21, 17, 13 and 11 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitate was observed at the start or end of the treatment period.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 µg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 – 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Treatment of cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37.0 ± 1.0°C in the presence of 5% CO2. In total 3 experiments were performed. In addition 4 experiments were rejected due to the fact that the variation in the vehicle controls was too high.

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

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption is measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.

Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x [(Vsample - Vblank) / (Vsolvent - V blank)]
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data intepretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
• The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

Experiment 1: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.98 and the EC1.5 20.3 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.85 and the EC1.5 38.1 µM.
Experiment 3: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.40 and the EC1.5 39.1 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Experiment 1 (Imax = 1.70 at 31.3 μM)

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (μM)

Value:

23

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Experiment 1 (IC30 = 43.1 μM)

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.62

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Experiment 2 (Imax = 1.62 at 32.8 μM). Luciferase activity induction (>1.5-fold compared to the vehicle control) only observed at a cytotoxic concentration (IC30 = 29.9 μM).

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: Imax

Value:

1.52

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Experiment 3 (Imax = 1.52 at 41.0 μM)

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 (μM)

Value:

40.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Experiment 3 (IC30 = 46.2 μM)

Other effects / acceptance of results:

In the first experiment, the test substance showed toxicity. The calculated IC30 was 43.1 μM and the calculated IC50 was 49.0 μM. A dose related luminesce activity induction was observed after treatment with the test item. The Imax was 1.70 and the EC1.5 23.0 μM. The dilution series for experiment 2 were adapted due to the toxicity observed and to carefully investigate the effect observed at 31.3 μM.
Toxicity was also seen in the second and third experiment. In the second experiment, the calculated IC30 was 29.9 μM and the calculated IC50 was 33.5 μM.
A luminesce activity induction compared to the vehicle control was only observed at 32.8 μM with an Imax of 1.62. Since the test item was toxic at this concentration (53.6% viability), no EC1.5 was calculated. In the third experiment, the calculated IC30 was 46.2 μM and the calculated IC50 was 47.7 μM. A dose related luminesce activity induction was observed after treatment with the test item. The Imax was 1.52 and the EC1.5 40.1 μM.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (17.8%, 15.1% and 9.8% in experiment 1, 2 and 3, respectively).
- Acceptance criteria met for positive control: yes. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 μM (20.3 μM, 38.1 μM and 39.1 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.98-fold, 2.85-fold and 2.40-fold in experiment 1, 2 and 3, respectively).

Table 1. Overview luminescence induction and cell viability of the test substance in Experiments 1, 2 and 3

Concentration (µM)	0.49	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000
Exp 1 luminescence	0.57	0.71	0.80	0.87	1.07	1.32	1.70***	0.00	0.00	0.00	0.00	0.26
Exp 1 viability (%)	81.2	80.6	73.8	87.3	80.0	92.4	110.6	3.7	3.4	4.4	1.5	5.7
Concentration (µM)	11	13	17	21	26	33	41	51	64	80	100	125
Exp 2 luminescence	0.86	0.88	1.13	1.29	1.22	1.62	0.65	0.27	0.00	0.00	0.00	0.00
Exp 2 viability (%)	82.2	77.8	77.7	78.6	91.3	53.6	10.3	14.1	0.0	-0.1	0.0	0.0
Exp 3 luminescence	0.74	0.76	0.90	1.02	1.11	1.32	1.52***	0.04	0.00	0.00	0.00	0.00
Exp 3 viability (%)	84.5	89.9	96.1	96.3	106.1	115.0	136.9	5.3	-1.0	-1.1	-1.0	-0.8

Table 2. Overview of luminescece induction and cell viability positive control Ethylene dimethacrylate glycol in Experiments 1, 2 and 3

Concentration (μM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.97	1.35	1.85***	2.32***	2.55***	3.98***
Exp 1 Viability (%)	108.3	93.0	109.0	113.7	107.8	99.0
Exp 2 luminescence	0.94	1.08	1.41	1.82***	2.31***	2.85***
Exp 2 Viability (%)	77.7	83.3	93.0	105.6	111.8	120.0
Exp 3 luminescence	0.97	1.19	1.41	1.77***	2.05***	2.40***
Exp 3 Viability (%)	73.1	87.8	89.9	127.0	124.4	116.3

Table 3. Overview of EC1.5, Imax, IC30 and IC50 values

	EC1.5 (μM)	Imax	IC30 (μM)	IC50 (μM)
Test item experiment 1	23.0	1.70	43.1	49.0
Test item experiment 2	NA	1.62	29.9	33.5
Test item experiment 3	40.1	1.52	46.2	47.7
Pos. control experiment 1	20.3	3.98	NA	NA
Pos. control experiment 2	38.1	2.85	NA	NA
Pos. control experiment 3	39.1	2.40	NA	NA

NA = Not applicable

Interpretation of results:

other: KeratinoSens assay was positive

Conclusions:

In a GLP-compliant guideline study, the test substance showed >1.5-fold induction at test concentrations of ≤1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. Based on this, the test substance is considered positive under the experimental conditions in this assay.

Executive summary:

A GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. In total three independent experiments were performed. The test substance was dissolved in dimethyl sulfoxide at 100 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 - 1000 μM (2-fold dilution series). The test item precipitated at the dose levels of 250, 500 and 1000 μM. Due to toxicity, the test item concentrations used for the second and third experiment were 11 - 125 μM (1.25-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The acceptance criteria were met in all three experiments. The test item showed toxicity (IC30 values of 43.1, 29.9 and 46.2 μM in experiment 1, 2 and 3, respectively and IC50 values of 49.0, 33.5 and 47.7 μM in experiment 1, 2 and 3, respectively).

In the first experiment, a statistically significant induction of the luciferase activity (EC1.5 value 23.0 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 1.70-fold at 31.3 μM. The test item was retested with a narrower dose response analysis using a lower dilution factor (1.25-fold) to determine if induction occurred again in a repeat experiment. In the second experiment, luciferase activity induction (>1.5-fold compared to the vehicle control) was only observed at a cytotoxic concentration (<70% viability compared to the vehicle control). Therefore this induction was not relevant and no EC1.5 was calculated. Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 40.1 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold at 41.0 μM. Overall, the test substance is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of ≤1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 February 2017 to 28 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E: In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

14 September 2015

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

Information from in vitro/in chemico test method(s) are preferred above the animal mouse LLNA study since 2016 according to Article 13(3) of regulation (EC) No 1907/2006. However, due to an inconclusive result from the in vitro/in chemico testing battery an LLNA has been performed as well.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Test item preparation: immediately prior to start a stable 1562 μg/mL test substance suspension in culture medium was prepared. Sonication for 5 minutes was used for the formulation of the stable suspension. Thereafter, the test item was further diluted to 78.1 μg/mL, the highest tested test item concentration in the h-CLAT runs.
The maximum concentration of test item tested in the XTT test was 312.5 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from the highest soluble concentration.
Medium Control and Solvent Control for the Test Item: Culture medium
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL)
Solvent Control for the Positive Control: DMSO (final concentration 0.2%)

Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers. The cell density should not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 11 in both XTT assays and 12, 17 and 23 in the h-CLAT for runs 1, 2 and 3, respectively.
Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells during the assay.
Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9- 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Dose finding assay: The doses investigated in the main experiment (h-CLAT) were determined with two XTT tests, instead of flow cytometry recommended by the guideline. 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. A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The CV75 could not be determined therefore the highest soluble test item concentration was used to calculate the dose-range for the main experiment (h-CLAT).

CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - [(Conc.>75 - Conc.<75) x (%>75 - 75)]/(%>75 - %<75), where:
a) Conc.>75 = maximal measured concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorpbance at a) in %
d) %<75 = relative absorpbance at b) in %

Treatment: 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 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations.
XTT Labelling and Measurement: 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 (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Acceptability criteria of XTT assay:
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test:
The test item was tested in three independent runs.
Test item preparation: Since the CV75 could not be determined, a stock solution of the highest soluble test item concentration was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 21.8, 26.2, 31.4, 37.7, 45.2, 54.2, 65.1 and 78.1 μg/mL.
Treatment of the cells: 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 ± 1 hours. Each concentration of the test item, medium control, positive and DMSO control was tested in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the cells: 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 procedure. The cells with the different antibodies or the IgG1were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
Sample preparation for measurement: 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-amino-actinomycin D (7-AAD) solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. 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.
Acquisition: A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).
Data analysis and interpretation:
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = 100 x [(MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells)], where MFI is geometric mean fluorescent intensity
The cell viability is calculated as follows:
Cell viability (%) = 100 x (Mean cytotoxicity of solvent control cells / Mean cytotoxicity of the test item treated cells), where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.
Acceptability criteria of the h-CLAT assay:
The study is considered as valid, if the following criteria are met:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test chemical, 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 < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 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 5000 μg/mL in saline (or medium or other solvents/vehicles), 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 > 90%.

Evaluation of results:
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is 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:
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, 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 without the need for a third run. If however, 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 will be 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 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered POSITIVE.

Positive control results:

The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI, CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

First run

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI, CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

First run

Key result

Run / experiment:

other: 2

Parameter:

other: % RFI, CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Second run

Key result

Run / experiment:

other: 2

Parameter:

other: % RFI, CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Second run

Key result

Run / experiment:

other: 3

Parameter:

other: % RFI, CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Third run

Key result

Run / experiment:

other: 3

Parameter:

other: % RFI, CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Third run

Other effects / acceptance of results:

XTT test:
Cytotoxic effects were observed in the highest tested concentration (312.5 μg/mL) in the microscopic evaluation. However, no cytotoxic effects were observed in the photometric evaluation following incubation with the test item up to the highest tested concentration (312.5 μg/mL). A CV75 value could not be calculated. Since precipitations were observed in the two highest tested test item concentrations of both XTT tests, 78.1 μg/mL of the test item was tested as highest concentration in the h-CLAT runs.
- Acceptance criteria met for medium control: mean absorbance of the medium control is ≥ 0.5
- Acceptance criteria met for solvent control: mean viability of the solvent control is ≥ 90% in comparison to the medium control
The XTT test is considered to be acceptable.

Main test:
The test item with a log Pow of 5.7 was tested in 3 independent runs. Precipitation was observed in the highest tested test item concentration (78.1 μg/mL) of the first h-CLAT run. Therefore, the result of this test item concentration was excluded from the evaluation.
The RFI of CD54 was greater than 200% in all concentrations of the first run. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the second and third run. Two out of three runs showed a NEGATIVE result. However, due to a log Pow > 3.5 for the tested test item, the result of this h-CLAT should not be considered.
The cell viability of the highest tested test item concentration was not < 90%. However, precipitation was observed in the dose range finding experiments (XTT tests) at concentrations above 78.1 μg/mL and precipitation was observed in the first h-CLAT run at 78.1 μg/mL. Therefore the highest soluble test item concentration was used as the maximal test concentration and a negative result is acceptable even if the cell viability is > 90%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
- Acceptance criteria met for positive control: 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%.
- Cell viability of the DMSO control was more than 90% in comparison to the medium control.
- For both medium and DMSO solvent controls, the MFI ratio of CD86 and CD54 to isotype control was > 105%.
- For the test chemical, the cell viability was more than 50% in at least four tested concentrations in each run.
All valid runs meet the acceptance criteria.

Results of the XTT assay:

1st XTT test:

		Microscopic Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.676	0.030	0.220	0.456	99.37
Solvent Control	-	no	0.677	0.030	0.218	0.459	100.00
Test Item	2.4	no	0.643	0.038	0.216	0.427	92.97
	4.9	no	0.685	0.038	0.217	0.468	101.91
	9.8	no	0.717	0.036	0.216	0.502	109.20
	19.5	no	0.777	0.029	0.219	0.558	121.41
	39.1	no	0.760	0.032	0.220	0.540	117.62
	78.1	no	0.782	0.035	0.220	0.562	122.37
	156.3^P	no	0.709	0.024	0.218	0.491	106.83
	312.5^P	yes	0.643	0.023	0.212	0.432	93.94

P         precipitation

*         mean absorbance (absolute) of 7 wells

**       relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 100.6%.

The CV75 value of the first XTT could not be calculated.

2nd XTT test:

		Microscopic Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.622	0.037	0.216	0.406	94.95
Solvent Control	-	no	0.631	0.020	0.204	0.428	100.00
Test Item	2.4	no	0.638	0.031	0.215	0.423	98.93
	4.9	no	0.704	0.033	0.210	0.494	115.45
	9.8	no	0.692	0.037	0.215	0.477	111.51
	19.5	no	0.740	0.055	0.215	0.525	122.67
	39.1	no	0.734	0.031	0.207	0.527	123.24
	78.1	no	0.665	0.026	0.208	0.457	106.78
	156.3^P	no	0.639	0.031	0.214	0.425	99.30
	312.5^P	yes	0.598	0.029	0.213	0.386	90.20

P         precipitation

*         mean absorbance (absolute) of 7 wells

**       relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 105.3%.

The CV75 value of the second XTT could not be calculated.

Due to the lack of cytotoxicity in both XTT tests, a CV75 value could not be calculated.

Results of the h-CLAT test:

1st test run:

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	328.7*	1175.2*	86.8
	3.0	520.7*	938.1*	74.8
Test Item	21.8	278.6*	106.3	98.1
	26.2	282.7*	86.0	97.1
	31.4	296.9*	84.6	94.7
	37.7	351.0*	109.1	98.3
	45.2	348.0*	91.6	95.9
	54.2	345.9*	101.4	95.8
	65.1	373.5*	114.7	93.1
	78.1^P	385.7*	132.9	93.0

P         Precipitation (will be excluded from the evaluation)

*         RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

2nd test run:

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	213.2*	392.0*	78.0
	3.0	200.4*	316.7*	61.0
Test Item	21.8	125.9	124.0	105.0
	26.2	122.4	117.8	99.7
	31.4	117.6	129.8	100.7
	37.7	116.9	107.0	99.3
	45.2	118.4	103.5	98.4
	54.2	123.9	111.6	98.4
	65.1	154.1	112.0	95.2
	78.1	158.4	104.7	97.8

*         RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

3rd test run:

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	299.1*	783.9*	76.0
	3.0	441.5*	700.8*	82.0
Test Item	21.8	102.3	86.5	96.9
	26.2	114.5	77.3	97.1
	31.4	96.2	75.5	84.8
	37.7	114.5	71.2	96.1
	45.2	104.6	73.6	98.1
	54.2	97.7	59.5	96.2
	65.1	114.5	68.7	94.2
	78.1	136.6	79.8	97.0

*         RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:

other: h-CLAT was inconclusive

Remarks:

because the Log Kow of the test substance is 5.7 and negative results for test chemicals with a Log Kow > 3.5 should not be considered.

Conclusions:

In a GLP-compliant guideline study, the test substance was found to be negative in the in vitro h-CLAT test. However, the negative result cannot be used in an assessment of skin sensitisation potential because the Log Kow of the test item is 5.7 and according to OECD test Guideline 442E, negative results for test chemicals with a Log Kow > 3.5 should not be considered. The h-CLAT is therefore considered inconclusive for the test substance.

Executive summary:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The validity criteria of the test were met, e.g. the values obtained for controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT) based on the results of two XTT tests: 21.8, 26.2, 31.4, 37.7, 45.2, 54.2, 65.1 and 78.1 µg/mL. The test substance was tested in 3 valid independent runs. Precipitation was observed in the highest tested test item concentration (78.1 μg/mL) of the first h-CLAT run. Therefore, the result of this test item concentration was excluded from the evaluation. The RFI of CD54 was greater than 200% in all concentrations of the first run. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the second and third run. Two out of three runs showed a negative result. However, due to a log Kow > 3.5 for the tested test item, the result of this h-CLAT should not be considered. The h-CLAT is therefore considered inconclusive for the test substance.

Reference 5

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential is assessed with in vivo and in vitro/in chemico testing and is shortly summarized below. Thereafter the executive summaries of the individual studies are presented.

Summary:

An LLNA study is conducted as the result of the in vitro/in chemico skin sensitisation testing battery was inconclusive and therefore not adequate for classification. In the LLNA the test item, tested up to the maximum attainable concentration of 25% w/w in acetone/olive oil 4:1, showed negative results. 

The in vitro/in chemico skin sensitisation testing battery included the DPRA, KeratinoSens and h-CLAT test. The DPRA was negative, the KeratinoSens was positive and the h-CLAT was considered inconclusive. Based on the two of three tests rule the overall result approach, the overall conclusion of the in vitro/in chemico testing battery is that the test substance presents an inconclusive result in accordance with Bauch et al., 2012. Therefore the outcome of the in vitro/in chemico testing battery was not adequate for classification.

In conclusion: The LLNA is the Key study as the in vitro/in chemico testing battery is inconclusive. Based on the test item, presenting negative results in the LLNA when tested up to the maximum attainable concentration of 25% w/w in acetone/olive oil 4:1, the substance can be considered a non-sensitiser.

 

Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

Key study: LLNA (OECD TG 429)

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay and GLP. Following a vehicle determination trial, the maximum attainable concentration was determined to be 25% in acetone/olive oil 4:1. The maximum attainable concentration was 25% because the substance is a solid and wetting/dilution is needed to apply the substance on the mouse ear. In a preliminary screening test no clinical signs of toxicity were noted at this maximum attainable concentration. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. At 5, 10 and 25% the substance showed SI values of 1.65, 1.55 and 2.37, respectively. The test item failed to produce a threefold or greater increase in 3HTdR incorporation compared to control values and is therefore considered to be a non-sensitiser under the conditions of the test.

DPRA (OECD TG 442C)

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance were successfully analysed by the validated DPRA analytical method. In the Cysteine reactivity assay, Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM test substance, 0.5 mM Cysteine. In the Lysine reactivity assay, Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in Lysine peptide stock solution. The final sample concentration was 25 mM test substance, 0.5 mM Lysine. The individual peptide depletion results for the test substance were -1.09% (Cysteine) and -1.58% (Lysine). The mean result is therefore considered zero. All analytical acceptance criteria of the test were met. No co-elution peaks were observed in either the Cysteine or Lysine assays. With effectively no depletion of either peptide in the presence of the test substance, the test substance was classified in the “no to minimal reactivity” class based on the DPRA prediction model and was thus considered to be negative in the DPRA.

KeratinoSens (OECD TG 442D)

A GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. In total three independent experiments were performed. The test substance was dissolved in dimethyl sulfoxide at 100 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 - 1000 μM (2-fold dilution series). The test item precipitated at the dose levels of 250, 500 and 1000 μM. Due to toxicity, the test item concentrations used for the second and third experiment were 11 - 125 μM (1.25-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The acceptance criteria were met in all three experiments. The test item showed toxicity (IC30 values of 43.1, 29.9 and 46.2 μM in experiment 1, 2 and 3, respectively and IC50 values of 49.0, 33.5 and 47.7 μM in experiment 1, 2 and 3, respectively).

In the first experiment, a statistically significant induction of the luciferase activity (EC1.5 value 23.0 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 1.70-fold at 31.3 μM. The test item was retested with a narrower dose response analysis using a lower dilution factor (1.25-fold) to determine if induction occurred again in a repeat experiment. In the second experiment, luciferase activity induction (>1.5-fold compared to the vehicle control) was only observed at a cytotoxic concentration (<70% viability compared to the vehicle control). Therefore this induction was not relevant and no EC1.5 was calculated. Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 40.1 μM; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold at 41.0 μM. Overall, the test substance is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of ≤1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments.

h-CLAT (OECD TG 442E)

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The validity criteria of the test were met, e.g. the values obtained for controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT) based on the results of two XTT tests: 21.8, 26.2, 31.4, 37.7, 45.2, 54.2, 65.1 and 78.1 µg/mL. The test substance was tested in 3 valid independent runs. Precipitation was observed in the highest tested test item concentration (78.1 μg/mL) of the first h-CLAT run. Therefore, the result of this test item concentration was excluded from the evaluation. The RFI of CD54 was greater than 200% in all concentrations of the first run. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the second and third run. Two out of three runs showed a negative result. However, due to a log Kow > 3.5 for the tested test item, the result of this h-CLAT should not be considered. The h-CLAT is therefore considered inconclusive for the test substance.

Justification for classification or non-classification

Based on the presented information the substance is not a skin sensitiser. Therefore classification of this substance for skin sensitisation is not warranted according to EU CLP Regulation (EC) No. 1272/2008 and its updates.