4-(dodecylthio)-4-methylpentan-2-one

Endpoint summary

Description of key information

The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

 

DPRA assay, OECD 442C, 2018, WoE:

GR-87-7596 (Scentaurus juicy) was non-reactive and classified into the minimal reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

 

Keratinosens, OECD 442D, 2018, WoE:

In all three repetitions, induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as sensitizer.

 

hCLAT, OECD 442e, 2018, WoE:

Test article GR-87-7596 produced a positive sensitization response for both CD86 and CD54 in THP-1 human monocytic cells in two of three independent main tests conducted. Therefore, this test article is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2.5.2018 and 4.5.2018

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

Principles of method if other than guideline:

Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 5 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP.

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Acetonitrile (i.e. the standard solvent of the SOP) could be used for preparation of test solutions.The test was run according to the final validated SOP published by ECVAM (Dbalm protocol 154).

BASIS OF THE METHOD
Skin sensitizing chemicals have the ability to covalently modify skin proteins or to be biotically or abiotically activated to become protein-reactive. Chemical-modified proteins are recognized by the immune system as foreign and trigger a specific T-cell mediated immune response.
A key step in the skin sensitization process is therefore the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. Based on this wellestablished toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins. Gerberick et al. therefore developed a peptide depletion assay using different
heptapeptides (later coined the DPRA or ‘direct peptide reactivity assay’) to assess a chemicals ability to react with and deplete a test peptide. Depletion is measured as the loss of the peptide signal as determined by HPLC-UV.

EXPERIMENTAL DESCRIPTION
Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

HPLC-Conditions:
▪ LC-System: Agilent 1100 Series (quat. low pressure gradient pump)
▪ Column: Zorbax SB-C18, 3μm, 2.1mm x 100mm
▪ DAD-Detector: 220nm
▪ Inj.Vol.: 7μl, Temp.: 30°C, Flow: 0.35ml/min
▪ Mobile Phase: ACN + 0.085% TFA / H2O + 1% TFA
▪ Gradient: 0min: 10% ACN / 90% H2O
10min: 25% ACN / 75% H2O
11min: 90% ACN / 10% H2O
13min: 90% ACN / 10% H2O
13.5min - 20min: 10% ACN / 90% H2O (conditioning)

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control
In each test Cinnamic aldehyde is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

Prediction Model
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.
The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing:
Proficiency testing at the Givaudan testing facility’.

Positive control results:

All the acceptance criteria were fulfilled for the positive control cinnamic aldehyde.

Cys-peptide depletion:
Average: 66.1
Standard deviation: 1.1
Coefficient of variation: .1.6

Lys-peptide depletion:
Average: 53.6
Standard deviation: 2.0
Coefficient of variation: .3.7

Key result

Parameter:

other: Average depletion Cys- and Lys-peptides

Value:

4.5 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The test substance gave 9% depletion of the Cys-peptide and 0 % depletion of the Lys-peptide. The average peptide depletion is 4.5%. This is below the threshold of 13.89%, and the substance is thus attributed to the “minimal” reactivity class, rating it as a non-sensitizer according to the DPRA prediction model.

Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (2.5 and 0.5 % SD, respectively). The co-elution controls indicated no co-elution with an UV-absorbing component.

Interpretation of results:

other: DPRA test result itself does not classify the substance as skin sensitizer.

Conclusions:

GR-87-7596 was non-reactive and classified into the minimal reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Executive summary:

Introduction

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides.

This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

Experimental

The test substance GR-87-7596 was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by GR-87-7596 was determined by HPLC-UV.

Results

GR-87-7596 was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 August to 06 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

The test article was tested for solubility in dimethyl sulfoxide (DMSO, at 500 mg/ml),
0.9% saline (at 100 mg/ml), and ethanol (EtOH, at 100 and 500 mg/ml). The test article was not soluble
in DMSO or saline but was soluble at 100 and 500 mg/ml in EtOH. Therefore, the Study Director, in
consultation with the Sponsor, chose EtOH as the vehicle for the test article.
Seven controls were included in the study. Two reference materials, mercaptobenzothiazole (MBT) and
isopropanol, were tested concurrently to confirm the utility of ethanol as a test article vehicle. The two
positive controls were 1-chloro-2,4-dinitrobenzene (DNCB, 4 μg/ml in DMSO) and nickel sulfate (NiSO4,
100 μg/ml in saline). The negative control was lactic acid (LA, 1000 μg/ml in saline). The vehicle
controls were EtOH (1% in RPMI-10 medium) for the test article, reference materials, DMSO (0.2% in
RPMI-10 medium) for DNCB, and saline (1% in RPMI-10 medium) for NiSO4 and LA. The RPMI-10
medium alone (100%) was also included as a control.
The assay was first conducted using only the controls, not the test article or reference materials, to check
the reactivity of the cells. THP-1 human monocytic cells were seeded in 24-well plates at a
concentration of approximately 1 x 106 cells in 0.5 ml of RPMI-10 medium. Cells were dosed at one well
per control and incubated for approximately 24 hours. The cells were then treated with propidium iodide
(PI), to determine viability, along with antibody stain to evaluate CD86 and CD54 expression.
Three independent viability screens were then conducted using the test article and the EtOH vehicle
control. Two viability screens were conducted for each of the reference materials, plus EtOH. In all
screens, THP-1 cells were seeded at approximately 1 x 106 cells in 0.5 ml of RPMI-10 culture medium
and dosed at one well per concentration of the test article or vehicle control. The cells were incubated for
approximately 24 hours and stained with propidium iodide. Eight concentrations of the test article or
reference material were tested. For the test article, the concentration at which cell viability was reduced
to 75% (CV75) could not be calculated in Screen 1; therefore, a lower range of test article concentrations
was tested in Screens 2 and 3 and CV75 values were calculated. The CV75 was calculated for each
screen treated with the MBT reference material. A CV75 for screens treated with the isopropanol
reference material could not be calculated because no concentration produced a cell viability less than
75%.

Three main tests, as independent assays, were then conducted in a similar manner as the reactivity
check for the test article and for each of the reference materials, each including all controls to determine
CD86 and CD54 expression. The test materials were each assayed at eight concentrations, either
bracketing the CV75 or using the maximum concentration (as per the guideline) in EtOH, with 1.2-fold
serial dilutions. For each treatment, flow cytometry analysis was used to measure cell viability and the
Mean Fluorescence Intensity (MFI) of the viable cells for isotype, CD86, and CD54. Relative
Fluorescence Intensity (RFI) values were calculated for each test article or reference material
concentration and each control versus the isotype control.
The effective concentration (EC) values (i.e., the concentration at which the test article induced a CD54 RFI
value of 200 or a CD86 RFI value of 150) was calculated for test article GR-87-7596 and for the MBT
reference material in all main tests in which a positive sensitization response occurred. EC150 and EC200
values could not be calculated for reference material isopropanol because no positive sensitization
response occurred in any of the main tests.

Vehicle / solvent control:

other: 1% Ethanol in RPMI-10 medium

Negative control:

DL-Lactic acid

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

For both the DNCB and the NiSO4 positive controls, the mean cell viabilities were greater than 50%, the CD86 RFI values were greater than 150, and the CD54 RFI values were greater than 200. The positive controls passed all acceptance criteria in each of the main tests.

Key result

Parameter:

other: RFI CD86 : EC 150

Value:

20.46 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Parameter:

other: RFI CD54 : EC 200

Value:

22.17 µg/mL

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 mean cell viabilities for all eight concentrations of test article GR-87-7596 were 77.6% to 95.0% in Main Test 1, 85.0% to 95.4% in Main Test 2, and 91.4% to 95.8% in Main Test 3. All tests met the acceptance criterion of a mean cell viability of 50% or more for at least four concentrations. Main Test 3, which showed a negative result, had a mean cell viability of 92.0% at 1.2x CV75. As per the guideline, negative results are acceptable only for test chemicals exhibiting cell viability at 1.2x CV75 of less than 90%.
However, since both Main Tests 1 and 2 showed positive results, these results outweigh the results of Main Test 3 whether or not the test is considered acceptable.

Based on the mean CV75 value calculated from the screens, eight concentrations (8.5, 10.2, 12.2, 14.7, 17.6, 21.1, 25.3, and 30.4 μg/ml) of test article GR-87-7596 in EtOH were tested in the main tests. These concentrations were serial dilutions of 1.2-fold (i.e., eight doses ranging from 0.335 times CV75 to
approximately 1.2 times CV75). The main test was conducted three times, in independent assays.
The test article produced a positive sensitization response in both CD86 and CD54 in Main Tests 1 and 2.
Due to a calculation error in Main Test 2, the results of this test were initially considered to be negative.
Because the results of the first two main tests apparently did not agree, a third main test was conducted, and the results were negative for both CD86 and CD54. However, the corrected calculation of RFI values for Main Test 2 showed a positive sensitization response in both CD86 and CD54. Therefore, as per
OECD Guideline 442E, the h-CLAT prediction was considered positive.

Interpretation of results:

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

Conclusions:

Test article GR-87-7596 produced a positive sensitization response for both CD86 and CD54 in THP-1 human monocytic cells in two of three independent main tests conducted. Therefore, this test article is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT). Effective concentration values were found to be 20.46 microg/ml for CD86 and 22.17 microg/ml for CD54.
Reference material mercaptobenzothiazole produced a positive sensitization response for CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted, but a negative response for CD86 in all main tests. Therefore, this reference material is considered a potential dermal sensitizer in
the Human Cell Line Activation Test (h-CLAT). The effective concentration value was 50.34 microg/ml for CD54.
Reference material isopropanol did not produce a positive sensitization response for either CD86 or CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted.
Therefore, this reference material is not considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT).

Executive summary:

To determine the skin sensitization potential of a test article due to cell activation by the measurement of changes in the expression of dendritic cell surface markers (CD86, CD54) via flow cytometry. The THP-1 human monocytic cell line serves as the test system. The h-CLAT is

recommended by EURL ECVAM as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between sensitizers (i.e., UN GHS Category 1) and non-sensitizers for the purpose of hazard classification and labeling, per OECD Guideline 442E “In Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT).”

The test article was tested for solubility in dimethyl sulfoxide (DMSO, at 500 mg/ml), 0.9% saline (at 100 mg/ml), and ethanol (EtOH, at 100 and 500 mg/ml). The test article was not soluble in DMSO or saline but was soluble at 100 and 500 mg/ml in EtOH. Therefore, the Study Director, in consultation with the Sponsor, chose EtOH as the vehicle for the test article.

Seven controls were included in the study. Two reference materials, mercaptobenzothiazole (MBT) and isopropanol, were tested concurrently to confirm the utility of ethanol as a test article vehicle. The two positive controls were 1-chloro-2,4-dinitrobenzene (DNCB, 4 μg/ml in DMSO) and nickel sulfate (NiSO4,

100 μg/ml in saline). The negative control was lactic acid (LA, 1000 μg/ml in saline). The vehicle controls were EtOH (1% in RPMI-10 medium) for the test article, reference materials, DMSO (0.2% in RPMI-10 medium) for DNCB, and saline (1% in RPMI-10 medium) for NiSO4 and LA. The RPMI-10 medium alone (100%) was also included as a control.

The assay was first conducted using only the controls, not the test article or reference materials, to check the reactivity of the cells. THP-1 human monocytic cells were seeded in 24-well plates at a concentration of approximately 1 x 106 cells in 0.5 ml of RPMI-10 medium. Cells were dosed at one well per control and incubated for approximately 24 hours. The cells were then treated with propidium iodide (PI), to determine viability, along with antibody stain to evaluate CD86 and CD54 expression.

Three independent viability screens were then conducted using the test article and the EtOH vehicle control. Two viability screens were conducted for each of the reference materials, plus EtOH. In all screens, THP-1 cells were seeded at approximately 1 x 106 cells in 0.5 ml of RPMI-10 culture medium and dosed at one well per concentration of the test article or vehicle control. The cells were incubated for approximately 24 hours and stained with propidium iodide. Eight concentrations of the test article or reference material were tested. For the test article, the concentration at which cell viability was reduced to 75% (CV75) could not be calculated in Screen 1; therefore, a lower range of test article concentrations was tested in Screens 2 and 3 and CV75 values were calculated. The CV75 was calculated for each screen treated with the MBT reference material. A CV75 for screens treated with the isopropanol reference material could not be calculated because no concentration produced a cell viability less than 75%.

Three main tests, as independent assays, were then conducted in a similar manner as the reactivity check for the test article and for each of the reference materials, each including all controls to determine CD86 and CD54 expression. The test materials were each assayed at eight concentrations, eitherbracketing the CV75 or using the maximum concentration (as per the guideline) in EtOH, with 1.2-fold serial dilutions. For each treatment, flow cytometry analysis was used to measure cell viability and the Mean Fluorescence Intensity (MFI) of the viable cells for isotype, CD86, and CD54. Relative Fluorescence Intensity (RFI) values were calculated for each test article or reference material concentration and each control versus the isotype control.

The effective concentration (EC) values (i.e., the concentration at which the test article induced a CD54 RFI value of 200 or a CD86 RFI value of 150) was calculated for test article GR-87-7596 and for the MBT reference material in all main tests in which a positive sensitization response occurred. EC150 and EC200

values could not be calculated for reference material isopropanol because no positive sensitization response occurred in any of the main tests.

Test article GR-87-7596 produced a positive sensitization response for both CD86 and CD54 in THP-1 human monocytic cells in two of three independent main tests conducted. Therefore, this test article is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT). Effective concentration values were found to be 20.46 microg/ml for CD86 and 22.17 microg/ml for CD54.

Reference material mercaptobenzothiazole produced a positive sensitization response for CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted, but a negative response for CD86 in all main tests. Therefore, this reference material is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT). The effective concentration value was 50.34 microg/ml for CD54.

Reference material isopropanol did not produce a positive sensitization response for either CD86 or CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted.

Therefore, this reference material is not considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2.4.2018 and 19.4.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 5 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP.

Type of study:

activation of keratinocytes

Details on the study design:

Positive control: Cinnamic aldehyde
Negative (vehicle) control: Dimethylsulfoxide

All test reagents were sourced as indicated in the standard operating procedure. Luciferin was sourced from Promega.
The Luciferase substrate was prepared according to the following recipe: 20 mM Tricine; 2.67 mM MgSO4; 0.1 mM EDTA; 33.3 mM DTT; 270 μM Coenzyme A; 470 μM Luciferin; 530 μM ATP; pH 7.8.

Experimental procedures:
DMSO (i.e. the standard solvent of the SOP) could be used for preparation of test solutions. The test was run according to the final validated SOP published by ECVAM (Dbalm protocol 155).

BASIS OF THE METHOD
The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential to be metabolically transformed to electrophilic chemicals. The signaling pathway with the repressor protein Keap1(Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE / EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular endpoint to detect skin sensitizers in vitro [10]. This result was confirmed by independent laboratories.

EXPERIMENTAL DESCRIPTION
Test System(s):
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene AKR1C2.
The KeratinoSens™ cell line was developed by the testing lab and stored on liquid nitrogen. It was grown in 10 cm petri dishes as described in the SOP to 80% confluency prior to testing for 3 – 4 days.
Cells were counted using a counting chamber and adjusted to the desired density. During seeding into 96-well plates, the cell suspension was gently stirred and cell sedimentation was avoided by repeatedly pipetting up and down to ensure homogeneous distribution of cells.

Basic Procedure:
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Positive control
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five concentrations from 4 – 64 μM.

Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test substances and (ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell batch and made up with the same dilutions of the test substances.
Luminescence was read in a Promega Glomax Luminometer programmed to
i. add 50 μl of the luciferase substrate to each well,
ii. to then wait for 1 second and
iii. then to integrate the luciferase activity for 2 seconds.

Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5-, 2- and 3- fold induction (EC1.5, EC2 and EC3) are calculated. For cytotoxicity the IC50 value is extrapolated.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into this template, and all data processing is performed automatically by this Excel sheet.
For both the MTT and the luciferase data, first the background value recorded in an empty well without added cells is subtracted.
For the MTT data the % viability is then calculated for each well in the test plate in relation to the average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well in the test plate the fold induction is calculated in relation to this value.
The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response
up to 1000 μM
• EC 1.5 Concentration in μM for 1.5-fold gene induction
• EC 2 Concentration in μM for 2-fold gene induction
• EC 3 Concentration in μM for 3-fold gene induction
• Pos / Neg Rating of substance according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in μM for 50% reduction of cell viability

Prediction Model
Substances are rated positive if the following conditions are met:
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above the solvent control in a particular repetition as determined by students T-test. The EC1.5 value is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to 1.5, the substance is still rated negative and no EC1.5 value is calculated by the evaluation sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

Testing of Proficiency chemicals and historical positive control data in the test facility
The KeratinoSens assay was originally developed at the testing facility. Data for the Proficiency chemicals as defined by OECD TG 442d generated in the laboratory are summarized in the Givaudan report RCR 153’464 ’KeratinoSens assay: Proficiency testing at the testing facility’.
The validation of the luciferase readings according to Annex 3 of the OECD guideline is also given in that report.
These validations show a very good reproducibility of the assay over time.

Positive control results:

Cinnamic aldehyde was run in all three repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This requirement was fulfilled in all three repetitions. The induction at 64 μM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are:
(i) Average induction in the three replicates for cinnamic aldehyde at 64 μM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 μM and 30 μM. At least one of these two numerical criteria must be met in order to accept a repetition. Both criteria were fulfilled in all three repetitions.
Thus all three repetitions were valid for the positive control.

Key result

Parameter:

other: EC 1.5 geometric mean

Value:

10.75 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: EC 2 geometric mean

Value:

19.28 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: EC 3 geometric mean

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no induction above a given treshold

Other effects / acceptance of results:

GR-87-7596-4 was significantly cytotoxic in the tested concentration range.
GR-87-7596-4 did induce the luciferase gene above the threshold of 1.5 in all three repetitions and is thus rated as a sensitizer in the KeratinoSens™ assay.

Interpretation of results:

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

Conclusions:

In all three repetitions, induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as sensitizer.

Executive summary:

Introduction

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.

This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

Experimental

The test substance GR-87-7596-4 was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

Results

GR-87-7596-4 was significantly toxic to the KeratinoSens™ cells. It did induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

DPRA

The Direct peptide reactivity Assay (DPRA) was conducted to determine the reactivity of the test substance towards peptides, in accordance with the OECD Guideline 442c.

This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by test substance was determined by HPLC-UV.

The test item was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model.

It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

 

KeratinoSens

The test substance was submitted to the KeratinoSensTM test which is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response, following the OECD test guideline 442d. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

The test substance was significantly toxic to the KeratinoSens™ cells. It did induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay.

 

Human Cell Line Activation Test (h-CLAT)

The objective of this study is to determine the skin sensitization potential of the test substance due to cell activation by the measurement of changes in the expression of dendritic cell surface markers (CD86, CD54) via flow cytometry. The THP-1 human monocytic cell line serves as the test system. The h-CLAT is recommended by EURL ECVAM as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between sensitizers (i.e., UN GHS Category 1) and non-sensitizers for the purpose of hazard classification and labeling, per OECD Guideline 442E “In Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT).

The test substance was soluble at 100 and 500 mg/ml in EtOH (choosen as the vehicle for the test). Seven controls were included in the study. Two reference materials, mercaptobenzothiazole (MBT) and isopropanol, were tested concurrently to confirm the utility of ethanol as a test article vehicle. The two positive controls were 1-chloro-2,4-dinitrobenzene (DNCB, 4 μg/ml in DMSO) and nickel sulfate (NiSO4, 100 μg/ml in saline). The negative control was lactic acid (LA, 1000 μg/ml in saline). The vehicle controls were EtOH (1% in RPMI-10 medium) for the test article, reference materials, DMSO (0.2% in RPMI-10 medium) for DNCB, and saline (1% in RPMI-10 medium) for NiSO4 and LA. The RPMI-10 medium alone (100%) was also included as a control. The assay was first conducted using only the controls, not the test article or reference materials, to check the reactivity of the cells. THP-1 human monocytic cells were seeded in 24-well plates at a concentration of approximately 1 x 106 cells in 0.5 ml of RPMI-10 medium. Cells were dosed at one well per control and incubated for approximately 24 hours. The cells were then treated with propidium iodide (PI), to determine viability, along with antibody stain to evaluate CD86 and CD54 expression. Three independent viability screens were then conducted using the test article and the EtOH vehicle control. Two viability screens were conducted for each of the reference materials, plus EtOH. In all screens, THP-1 cells were seeded at approximately 1 x 106 cells in 0.5 ml of RPMI-10 culture medium and dosed at one well per concentration of the test article or vehicle control. The cells were incubated for approximately 24 hours and stained with propidium iodide. Eight concentrations of the test article or reference material were tested. For the test article, the concentration at which cell viability was reduced to 75% (CV75) could not be calculated in Screen 1; therefore, a lower range of test article concentrations was tested in Screens 2 and 3 and CV75 values were calculated. The CV75 was calculated for each screen treated with the MBT reference material. A CV75 for screens treated with the isopropanol reference material could not be calculated because no concentration produced a cell viability less than 75%.

Three main tests, as independent assays, were then conducted in a similar manner as the reactivity check for the test article and for each of the reference materials, each including all controls to determine CD86 and CD54 expression. The test materials were each assayed at eight concentrations, either bracketing the CV75 or using the maximum concentration (as per the guideline) in EtOH, with 1.2-fold serial dilutions. For each treatment, flow cytometry analysis was used to measure cell viability and the Mean Fluorescence Intensity (MFI) of the viable cells for isotype, CD86, and CD54. Relative Fluorescence Intensity (RFI) values were calculated for each test article or reference material concentration and each control versus the isotype control. The effective concentration (EC) values (i.e., the concentration at which the test article induced a CD54 RFI value of 200 or a CD86 RFI value of 150) was calculated for test substance and for the MBT reference material in all main tests in which a positive sensitization response occurred. EC150 and EC200 values could not be calculated for reference material isopropanol because no positive sensitization response occurred in any of the main tests.

The test substance produced a positive sensitization response for both CD86 and CD54 in THP-1 human monocytic cells in two of three independent main tests conducted. Therefore, this test substance is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT). Effective concentration values were found to be 20.46 μg/ml for CD86 and 22.17 μg/ml for CD54. Reference material mercaptobenzothiazole produced a positive sensitization response for CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted, but a negative response for CD86 in all main tests. Therefore, this reference material is considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT). The effective concentration value was 50.34 μg/ml for CD54. Reference material isopropanol did not produce a positive sensitization response for either CD86 or CD54 in THP-1 human monocytic cells in three of three valid independent main tests conducted. Therefore, this reference material is not considered a potential dermal sensitizer in the Human Cell Line Activation Test (h-CLAT).

Justification for classification or non-classification

Based on the available data, the substance is classified as skin sensitiser in Category 1B (H317: May cause an allergic skin reaction) according to the CLP and to the GHS.

 

No data was available for respiratory sensitisation.