1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

Endpoint summary

Description of key information

Weight of Evidence: skin sensitising

1. Molecular initiating Key Event 1: negative, DPRA, OECD TG 442C, 2021
Cysteine reactivity assay : (mean ; n=3): 0.58% ± 0.72% ; negative : No or minimal reactivity.
Lysine reactivity assay : (mean ; n=3): 0.19% ± 0.28% ; negative : No or minimal reactivity.
There were acceptability criteria exceptions (LYS-PC result and/or mean Cysteine peptide concentration of the Reference Control C)
The result will be considered within a weight of evidence for assessment for purposes of classification and labelling

2. Molecular initiating Key Event 2: positive (LC induction: > 1.5 µM observed in 3 of 3 experiments and up to 2000 μM) (n=3 replicates), KeratinoSens, OECD TG 442D, 2022

3. Molecular initiating Key Event 3: No data

4. QSAR : Skin sensitization GHS v.02.04 : potential for sensitisation - GHS Skin Sensitiser 1B (weak sensitiser) based upon potential for formation of (n=1) GHS Skin Sensitiser 1B metabolite. No other metabolites are predicted to be sensitisers, (ACF: 50% correct, 0% incorrect, 50% unknown), OASIS TIMES v2.31.2.82, 2022

 

Conclusion: Within the battery of in silico modelling and in vitro test assays, there are equivocal to positive predictions for sensitisation. The test item appears to be either a non-sensitiser to weak sensitiser. Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. (GHS Classification: Skin Sensitisation Category 1B).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

(Q)SAR predictions from OASIS TIMES v2.31.2.82 using the submodel: Skin sensitization GHS v.02.04. The prediction will be considered within a weight of evidence for assessment for Classification and Labelling purposes.

Justification for type of information:

1. SOFTWARE
Skin sensitization GHS v.02.04; within OASIS TIMES v2.31.2
Contact LMC University:
Prof. As. Zlatarov,
LMC University,
Laboratory of Mathematical Chemistry,
Bourgas,
Bulgaria
URL: www.oasis-lmc.org
More information available at:
http://oasis-lmc.org/products/models/human-health-endpoints/skin-sensitization.aspx

2. MODEL (incl. version number)
OASIS TIMES v2.31.2 model v.02.04, Skin sensitization GHS
March 2017 (model development and/or QMRF publication) ; January 2020 (previous model version) ; November 2021 (current model version).

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See attached: ‘OASIS TIMES v.2.31.2.82 Prediction on Skin Sensitisation of [test item name] (GHS v.02.04)’ version 1.0; dated 12 May 2022.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Full details of the method are provided in the attached QMRF named ‘Skin sensitization GHS model’ for OASIS TIMES v2.31.2 model v.02.04, Skin sensitization GHS ; version 1.5; 01 November 2021

5. APPLICABILITY DOMAIN
See ‘any other information on results incl. tables’.
See attached: ‘OASIS TIMES v.2.31.2.82 Prediction on Skin Sensitisation of [test item name] (GHS v.02.04) ’ version 1.0; dated 12 May 2022.
- Descriptor/Parameter domain: All constituents are in domain.
- Structural and mechanistic domains: Correct fragments = 50%, Incorrect fragments = 0%, Unknown fragments = 50%, Total Structural domain : (Out of domain - 2 chemicals ; represented by generic SMILEs for both geometric isomers). All constituents are within the mechanistic domain.

6. ADEQUACY OF THE RESULT
1) QSAR model is scientifically valid. 2) The substance falls within the (general) applicability domain of the QSAR model and/or with additional interpretation by expert judgement. 3) The prediction is fit for regulatory purpose. Specifically when combined with further information such as physico-chemistry and in chemico and in vitro testing and further modelling. The result will be considered within a weight of evidence assessment for C&L and risk assessment purposes.

Guideline:

other: REACH Guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

Full details of the method are provided in the attached QMRF named ‘Skin sensitization GHS model’ - model: Skin sensitization GHS v.02.04 - software package: OASIS TIMES v2.31.2, version 1.5; date: 01 March 2017; updated 01 November 2021.
- The model applies the following methodology to generate predictions:
The LMC stepwise approach was used to define the applicability domain. It contains three layers:
(i) General properties requirements (log KOW, MW, log WS)
(ii) Structural domain (Atom Cantered Fragments (ACFs))
(iii) Metabolic domain
Then assessment of interpolation space - estimates the population density of the parametric space defined by the explanatory variables of the QSAR models by making use the training set chemicals
Details could be found in:
Dimitrov S, Dimitrova G., Pavlov T., Dimitrova D., Patlevisz G., Niemela J., Mekenyan O., A stepwise approach for defining the applicability domain of SAR and QSAR models, J. Chem. Inf. Model., 45, 839-849 (2005)
- The model and the training sets are collated and published by Prof. As. Zlatarov, LMC University, (Bulgaria).
The training set of the model currently consists of 1168 chemicals (proprietary)
The experimental values in the training set and validation set were measured using one or more methods equivalent or similar to the following guidelines:
- Local Lymph Node Assay (LLNA) eq. or similar to OECD TG 429
- Human Repeat Insult Patch Test (HRIPT).
- Other appropriately documented data, (collated by the model authors).
All available skin sensitization data (EC3,% and NOEL values in μg/cm2) from both tests were stored in the training set. In case of multiple data the worst case scenario (or expert judgement) is applied. Most potent data (EC3, NOEL) are selected to represent the individual training set chemical(s).
A full list of experimental reference citations is provided in the OASIS TIMES software with additional reference citations in the QMRF attached.
- Justification of QSAR prediction: The result should be considered in relation to corresponding information presented and in accordance with the tonnage driven information requirements of REACH Regulation (EC) 1907/2006 in a weight of evidence. For classification purposes, Regulation (EC) No 1272/2008 allows the use of non-standard approaches as mentioned in 4.1.1.2.2. Annex XI, 1.3 allows the use of a (Q)SAR instead of testing when certain conditions are met (use of valid (Q)SAR, adequate and reliable information is provided etc). The present predictions are intended to be used in a weight of evidence approach as mentioned in Annex IX, 1.2: using the present predictions to support experimental data (GLP and/or non-GLP ; completed to applicable guidelines and/or using methods which are scientifically validated and/or from reports that are sufficiently documented).

Specific details on test material used for the study:

Detailed information on the 'test material identity' is provided in the attached QSAR Prediction Reporting Format (QPRF) document including information on individual constituents.

Key result

Parameter:

other: GHS Skin Sensitiser sub-category

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Metabolite #4: Alert reliability: High, >= 60% (n>=5) ; Predicted skin sensitisation potency: GHS sub-category 1B Conclusion: based on in silico battery screening: there is potential for formation of (n=1) : GHS Skin Sensitiser 1B metabolite ; details of alert provided in attached QPRF ; no other metabolites are predicted to be sensitisers. Consequentially, based on weight of evidence and/or based on precautionary principle: substance is predicted to be GHS Skin Sensitiser sub-category 1B

1. Defined Endpoint:
QMRF 4. Human health effects
QMRF 4.6 Skin sensitisation
Reference to type of model used and description of results:
Skin sensitization GHS v.02.04, November 2021 (current model version)
Platform version: OASIS TIMES v2.31.2.82

2. Description of results and assessment of reliability of the prediction:
Results:
The parent chemical is a mixture or multi-constituent substance :
Represented by generic PARENT SMILES: CC(CCc1ccc(OC)cc1)=COCCc1ccccc1
Related geometric isomers are considered incorporated into the above and/or represented by the PARENT SMILES.
Full details of the constituents are provided in the attached QPRF: ‘OASIS TIMES v.2.31.2.82 Prediction on Skin Sensitisation of [test item name] (GHS v.02.04)’ version 1.0; dated 12 May 2022.
Inputs Used for Test Item: All modelling was based on input of SMILES notation for the test item
Alert / result: Metabolite #4: Alert reliability: High, >= 60% (n>=5) ; Predicted skin sensitisation potency: GHS sub-category 1B
Conclusion: based on in silico battery screening: there is potential for formation of (n=1) : GHS Skin Sensitiser 1B metabolite ; details of alert provided in attached QPRF ; no other metabolites are predicted to be sensitisers. Consequentially, based on weight of evidence and/or based on precautionary principle: substance is predicted to be GHS Skin Sensitiser sub-category 1B.

3. Uncertainty of the prediction and mechanistic domain:
The training set is embedded in the software of the model; refer to the QMRF which is available in OASIS TIMES v.2.31.2.82. The training set data is proprietary and not made publicly available by the model developers. It is noted that the software model itself determines if the test item falls within the general properties requirements [lipophilicity (log KOW), molecular weight (MW) and/or water solubility (WS)] domain; then the appropriate structural and mechanistic/metabolic domains. Full details are provided of the methodology in the corresponding QMRF for model OASIS TIMES v.2.31.2, Skin sensitization GHS v.02.04, November 2021. Interested parties are invited to contact the model author.

Uncertainty in the prediction relates to:
Statistical characteristics are provided (i) for the prediction: in the attached QPRF and (ii) for the model: in the attached QMRF, respectively. No further comments on the uncertainty of the prediction are provided by the author of the present QPRF. A summary of provided information is presented by the applicant in attachment with relevant citations. It is noted that the (Q)SAR prediction will not be used as a standalone prediction. The prediction is fit for regulatory purpose when combined with further information such as physico-chemistry and in chemico and in vitro testing and further modelling. The result will be considered within a weight of evidence assessment for C&L and risk assessment purposes.

Interpretation of results:

other: The test item gave a positive prediction for skin sensitisation GHS Skin Sensitiser sub-category 1B. The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

The results are adequate for the regulatory purpose.

Executive summary:

OASIS TIMES v2.31.2, Skin sensitization GHS v.02.04
Results:
The parent chemical is a mixture or multi-constituent substance
Represented by generic PARENT SMILES : GHS Skin Sensitiser 1B
Conclusion: The substance is expected to be a skin sensitiser. Based on in silico battery screening: there is potential for formation of (n=1) : GHS Skin Sensitiser 1B metabolite. No other metabolites are predicted to be sensitisers. Consequentially, based on weight of evidence and/or based on precautionary principle: substance is predicted to be GHS Skin Sensitiser sub-category 1B.

Adequacy of the QSAR:
1) QSAR model is scientifically valid. 2) The substance falls within the (general) applicability domain of the QSAR model and/or with additional interpretation by expert judgement. 3) The prediction is fit for regulatory purpose. Specifically when combined with further information such as physico-chemistry and in chemico and in vitro testing and further modelling. The result will be considered within a weight of evidence assessment for C&L and risk assessment purposes.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10-06-2020 to 05-01-2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017). The result will be considered within a weight of evidence for assessment for Classification and Labelling purposes.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Acceptability criteria not met for mean Lys peptide depletion of positive control and Cys peptide concentration of ref controls A&C are outside range: see 'other effects/acceptance of results' + 'Applicant's summary and conclusions' for more information.

GLP compliance:

yes

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (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 in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. For further information contact the test facility.
- HPLC-PDA (UV) methodology are reported in the full study report.

PREPARATION OF TEST SOLUTIONS
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. The test item was found to be soluble at a concentration of 100 mM, in acetonitrile (ACN) with vortex mixing for 1 minute. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in the study. The physical descriptions of the test item was noted to be “clear colourless non-viscous solution” immediately after preparation of the dilution. The test item, positive control and peptide samples were typically prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the peptide/derivative stock solutions: [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (e.g. ca. 0.501 mg SPCC/mL) was prepared by dissolving an appropriate amount of SPCC in phosphate buffer pH 7.5. The mixture was gently mixed on a shaker.
2. Lysine: A stock solution of 0.667 mM SPCL (e.g. ca. 0.518 mg SPCL/mL) was prepared by dissolving an appropriate amount of SPCL in of acetate buffer pH 10.1 followed by The mixture was gently mixed on a shaker
Peptide standards from stock solutions:
For each peptide (SPCC and SPCL) a set of serially diluted standards were prepared. The highest stock concentration of the standards (0.534 mM) was prepared in acetonitrile from the 0.667 mM peptide stock solutions. The remaining standards were prepared by a 1:1 serial dilution in dilution buffer (20% acetonitrile in phosphate buffer for the cysteine peptide or acetate buffer for the lysine peptide). Six standards were prepared at concentrations of 0.534 to 0.017 mM. A seventh standard was prepared containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the appropriate pre-labelled glass autosampler vials.
- Preparation of the test chemical solutions: For both the cysteine and lysine reactivity assay test item was pre-weighed into a glass vial and dissolved, just before use, in ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of a clear colourless non-viscous solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared typically less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the positive controls, reference controls and co-elution controls:
1. Cysteine: SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in glass vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and/or 50 µL peptide standard. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The means of Reference Control samples A and C were not both within the acceptance criteria of 0.50 ± 0.05 mM. Specifically, 1) the mean Cysteine peptide concentration of the Reference Control C (Acetonitrile) was 0.4497 mM and Reference Control C (Water) was 0.4440 mM, which is outside of the range of 0.45 to 0.55 mM. It was considered within the study that this was an acceptable deviation based on the totality of information. Specifically, the peak area Coefficient of Variation (CV) was <5% for both Reference Control C-Acetonitrile and -Water, which is well within the acceptable range of <15%. Reference Control C is used in the calculation to determine percent peptide depletion of the test article mixed with peptide. Additionally, all the acceptance criteria for Reference control A and B and standard curve R2 value were met. Considering this information in totality, marginally lower mean Cysteine peptide concentration. It was considered this would have a negligible impact on sensitisation prediction in Cysteine reactivity model. Furthermore, the concurrent positive control cinnamic aldehyde had a Cysteine peptide depletion of 60.85%, meeting the assay acceptance criteria.
2. Lysine: SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in glass vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL peptide standard. The SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM.
3. Positive controls: PC samples were prepared from 750 μL ’stock solution’ of 0.667 mM SPCC or SPCL (as applicable), 50 μL or 250 μL for Cysteine and Lysine PC (cinnamic aldehyde solution) at 100 mM in ACN and in case of Cysteine, only further 200 μL ACN.

INCUBATION
- Incubation conditions: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at room temperature (typically: ca. 25°C). The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24±2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 26 hours. Prior to HPLC analysis the samples were visually inspected for precipitation upon mixing with peptide. With cysteine the test item was “clear colourless non-viscous solution”. With lysine this was “cloudy white droplets with clear colourless non-viscous solution”. After the incubation and the HPLC run, the test item solutions were: cysteine: “clear colourless non-viscous solution” and/or “cloudy white droplets with clear colourless non-viscous solution”.
- Precipitation noted: Within the study there was no reports of precipitation (i.e. full phase separation).

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Yes. HPLC-PDA (UV) methodology are reported in the full study report. The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C using standard formula (see: OECD TG 442C).
- Verification of the suitability of the HPLC for test chemical and control substances: A gradient elution was used in this assay. The mobile phase changed from 10-25% acetonitrile over a 10 minute period to allow for optimal separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections. See ‘Acceptability criteria’ for further information. There were no reports of oxidation or dimerization within the test system, reported in the study.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: See above. An overview of the retention time and peak areas at 220 nm for both the Cysteine Reactivity Assay and Lysine Reactivity Assay, are presented in the full study report.

- ACCEPTABILITY CRITERIA:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9995 and SPLC r2 = 0.9999)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 60.85% ± 0.26% and SPCL 23.86% ± 2.81%) It was noted within the study, for Lysine peptide-positive control data of the peptide depletion of: 23.86%, which is below the lower bound threshold of 40.2%. Whereas the standard deviation for three replicates of positive control cinnamic aldehyde was 2.81, which is well within the acceptable range of <11.6. All the acceptance criteria for Reference control A, B and C and standard curve R2 value were met. It was considered, within the study that underprediction of positive control cinnamic aldehyde % Lysine peptide depletion should not impact the sensitisation potential prediction of the test item because regardless of Lysine data, as the test item is predicted to be potential non-sensitiser based on mean Cysteine only prediction model. It is noted by the applicant that this study will be considered within a weight of evidence assessment for C&L purposes.
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.26% and SPCL PC : SD = 2.81%). See comments above relating to the positive control replicates in Lysine assay.
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. The means of Reference Control samples A and C were not both within the acceptance criteria of 0.50 ± 0.05 mM. Specifically, 1) the mean Cysteine peptide concentration of the Reference Control C (Acetonitrile) was 0.4497 mM and Reference Control C (Water) was 0.4440 mM, which is outside of the range of 0.45 to 0.55 mM. It was considered within the study that this was an acceptable deviation based on the totality of information. Specifically, the peak area Coefficient of Variation (CV) was <5% for both Reference Control C-Acetonitrile and -Water, which is well within the acceptable range of <15%. Reference Control C is used in the calculation to determine percent peptide depletion of the test article mixed with peptide. Additionally, all the acceptance criteria for Reference control A and B and standard curve R2 value were met. Considering this information in totality, marginally lower mean Cysteine peptide concentration. It was considered this would have a negligible impact on sensitisation prediction in Cysteine reactivity model. Furthermore, the concurrent positive control cinnamic aldehyde had a Cysteine peptide depletion of 60.85%, meeting the assay acceptance criteria.
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.01 to 0.02% ; Lysine: Reference Controls B and C was 0.01%)
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 98.9%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile (ACN)
Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

- PC CYS-peptide depletion (mean): 60.85% ± 0.26% (high reactivity)
- PC LYS-peptide depletion (mean): 23.86% ± 2.81% (high reactivity)
It was noted within the study, for Lysine peptide-positive control data of the peptide depletion of: 23.86%, which is below the lower bound threshold of 40.2%. Whereas the standard deviation for three replicates of positive control cinnamic aldehyde was 2.81, which is well within the acceptable range of <11.6. All the acceptance criteria for Reference control A, B and C and standard curve R2 value were met. It was considered, within the study that underprediction of positive control cinnamic aldehyde % Lysine peptide depletion should not impact the sensitisation potential prediction of the test item because regardless of Lysine data, as the test item is predicted to be potential non-sensitiser based on mean Cysteine only prediction model. It is noted by the applicant that this study will be considered within a weight of evidence assessment for C&L purposes.

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Remarks:

Mean (%) Cys-peptide depletion

Value:

0.58

Vehicle controls validity:

valid

Remarks:

See comments in "Other effects/acceptance of results"

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: (mean ; n=3): 0.58% ± 0.72% ; negative : No or minimal reactivity

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Remarks:

Mean (%) Lys-peptide depletion

Value:

0.19

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

other: See comments in "Other effects/acceptance of results"

Remarks:

See comments in 'Other effects / acceptance of results'

Remarks on result:

other: (mean ; n=2): 0.19% ± 0.28% ; negative : No or minimal reactivity

Outcome of the prediction model:

other: See comments provided below in 'Any other information on results incl. tables' and/or 'Applicant's summary and conclusion'

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.
- Other: Dilutions of test item and peptides were visually inspected for precipitation / phase separation immediately after mixing test items with peptides and after the HPLC run. The test item dilution with lysine was described “cloudy white droplets with clear colourless non-viscous solution” after mixing and HPLC run. With cysteine the test item was “clear colourless non-viscous solution” after mixing and HPLC run. Within the study there was no reports of precipitation (i.e. full phase separation), but observations with lysine may affect the results.

DEMONSTRATION OF TECHNICAL PROFICIENCY: 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 in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. For further information contact the test facility.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met with exceptions. See comments provided in (iv) below.
- Acceptance criteria met for positive control: All criteria met with exceptions. See comments provided in (ii) and (iii) below. It is noted by the applicant that this study will be considered within a weight of evidence assessment for C&L purposes.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9995 and SPLC r2 = 0.9999)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 60.85% ± 0.26% and SPCL 23.86% ± 2.81%). It was noted within the study, for Lysine peptide-positive control data of the peptide depletion of: 23.86%, which is below the lower bound threshold of 40.2%. Whereas the standard deviation for three replicates of positive control cinnamic aldehyde was 2.81, which is well within the acceptable range of <11.6. All the acceptance criteria for Reference control A, B and C and standard curve R2 value were met. It was considered, within the study that underprediction of positive control cinnamic aldehyde % Lysine peptide depletion should not impact the sensitisation potential prediction of the test item because regardless of Lysine data, as the test item is predicted to be potential non-sensitiser based on mean Cysteine only prediction model. It is noted by the applicant that this study will be considered within a weight of evidence assessment for C&L purposes.
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.26% and SPCL PC : SD = 2.81%). See comments above relating to the positive control replicates in Lysine assay.
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. The means of Reference Control samples A and C were not both within the acceptance criteria of 0.50 ± 0.05 mM. Specifically, 1) the mean Cysteine peptide concentration of the Reference Control C (Acetonitrile) was 0.4497 mM and Reference Control C (Water) was 0.4440 mM, which is outside of the range of 0.45 to 0.55 mM. It was considered within the study that this was an acceptable deviation based on the totality of information. Specifically, the peak area Coefficient of Variation (CV) was <5% for both Reference Control C-Acetonitrile and -Water, which is well within the acceptable range of <15%. Reference Control C is used in the calculation to determine percent peptide depletion of the test article mixed with peptide. Additionally, all the acceptance criteria for Reference control A and B and standard curve R2 value were met. Considering this information in totality, marginally lower mean Cysteine peptide concentration. It was considered this would have a negligible impact on sensitisation prediction in Cysteine reactivity model. Furthermore, the concurrent positive control cinnamic aldehyde had a Cysteine peptide depletion of 60.85%, meeting the assay acceptance criteria.
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.01 to 0.02% ; Lysine: Reference Controls B and C was 0.01%)

 Table 1.0 – Results of the DPRA with the test item

	SPCC depletion (CYSTEINE)		SPCL depletion (LYSINE)		Mean of SPCC and SPCL depletion
	Mean	± SD	Mean	± SD
Test item	0.58%	±0.72%	0.19%	±0.28%	0.39%

Conclusion:
The test item gave a negative in the DPRA. The test item was classified into a ‘no or minimal reactivity’ class using the Cysteine 1:10 / Lysine 1:50 prediction model.
Note: due to the acceptability criteria exceptions (see above) this study will be considered within a weight of evidence assessment for C&L purposes.

Interpretation of results:

other: The test item gave a negative in the DPRA. The test item was classified into a ‘no or minimal reactivity’ class using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for C&L purposes.

Conclusions:

The test item gave a negative in the DPRA.
The Mean of SPCC and SPCL depletion was 0.39%. Since the mean depletion was 0 to 6.38% the test item is predicted using the Cysteine 1:10 / Lysine 1:50 prediction mode to be in the ‘no to minimal’ reactivity class.

Executive summary:

The study was performed to the OECD TG 442C in chemico Direct Peptide Reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. The acceptance criteria were as follows: (i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9995 and SPLC r2 = 0.9999). (ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 60.85% ± 0.26% and SPCL 23.86% ± 2.81%) and/or (iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.26% and SPCL PC : SD = 2.81%). It was noted within the study, for Lysine peptide-positive control data of the peptide depletion of: 23.86%, which is below the lower bound threshold of 40.2%. Whereas the standard deviation for three replicates of positive control cinnamic aldehyde was 2.81, which is well within the acceptable range of <11.6. All the acceptance criteria for Reference control A, B and C and standard curve R2 value were met. It was considered, within the study that underprediction of positive control cinnamic aldehyde % Lysine peptide depletion should not impact the sensitisation potential prediction of the test item because regardless of Lysine data, as the test item is predicted to be potential non-sensitiser based on mean Cysteine only prediction model. (iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. The means of Reference Control samples A and C were not both within the acceptance criteria of 0.50 ± 0.05 mM. Specifically, 1) the mean Cysteine peptide concentration of the Reference Control C (Acetonitrile) was 0.4497 mM and Reference Control C (Water) was 0.4440 mM, which is outside of the range of 0.45 to 0.55 mM. It was considered within the study that this was an acceptable deviation based on the totality of information. Specifically, the peak area Coefficient of Variation (CV) was <5% for both Reference Control C-Acetonitrile and -Water, which is well within the acceptable range of <15%. Reference Control C is used in the calculation to determine percent peptide depletion of the test article mixed with peptide. Additionally, all the acceptance criteria for Reference control A and B and standard curve R2 value were met. Considering this information in totality, marginally lower mean Cysteine peptide concentration. It was considered this would have a negligible impact on sensitisation prediction in Cysteine reactivity model. Furthermore, the concurrent positive control cinnamic aldehyde had a Cysteine peptide depletion of 60.85%, meeting the assay acceptance criteria. (v) the  Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.01 to 0.02% ; Lysine: Reference Controls B and C was 0.01%). 

In the cysteine reactivity assay the test item showed 0.58% SPCC depletion while in the lysine reactivity assay the test item showed 0.19% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.39% and as a result the test item was considered to be in the Cysteine 1:10 / Lysine 1:50 prediction model 'no or minimal reactivity' class.

Within the study there was no reports of precipitation (i.e. full phase separation), but observations of cloudy white droplets with clear colourless non-viscous solution in the case of Lysine peptide with test item dilution may have affected the results.

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11-03-2020 to 02-05-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met . Test method considered to cover Key Event-2 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In test 3, the MTT viability incubation was for 22 minutes rather than 4 ± 0.3 hrs. This was not found to have adversely affected the study. Test 1,2 and 3 resulted in similar MTT viability response and/or overall predictions for test item and controls.

GLP compliance:

yes

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422D – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method and/or consistent with EURL ECVAM DB-ALM Protocol no. 155: KeratinoSens™, (Adopted March, 2018). The test facility participated within the ring trial for the adoption of the assay. Reference: Natsch et al., (2011). The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories. Toxicology in Vitro. 25(3) : 733-774.
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. For further information contact the test facility.

PREPARATION OF TEST SOLUTIONS
- Other: A solubility test was performed. The test item was suspended in DMSO at the highest x100 concentration of 2000 μM. The test item was found to be soluble in DMSO and formed a ‘clear colourless (non-viscous) solution’. DMSO was then utilised to further dilute test items within the definitive trials. No precipitation was reported during solubility trials. Prior to definitive testing, The ability of the test item to directly reduce MTT was assessed. Approximately 100 μL of the highest concentration of the x100 test item dilutions in DMSO were added to 1 mL of a 1.0 mg/mL MTT solution and then incubated in the dark at standard culture conditions (37 ± 1 °C humidified air containing 5 ± 1% CO2) for one hour. One hundred μL of the DMSO solvent control in 1 mL of a 1.0 mg/mL MTT solution was tested concurrently. If the MTT solution colour turned blue/purple, the test article was presumed to have reduced the MTT. Within the study, the test item was observed to directly reduce MTT.
- Preparation of the test chemical stock solution: In the main experiments the test item was dissolved in DMSO at the highest x100 concentration of 2000 μM. From this the test item solution was diluted to final x1 concentrations in 1% DMEM exposure medium. Resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM.
- Preparation of the test chemical serial dilutions: See above.
- Preparation of the positive controls: The positive control was Cinnamic aldehyde. The positive control was initially dissolved and diluted in DMSO to prepare a 64,000 μM dilution, which was further diluted 1:10 in DMSO to prepare a 6400 μM 100X stock concentration. The final x1 concentrations of the positive control were 64, 32, 16, 8, and 4 μM prepared in 1% DMEM (DMEM containing 1% FBS). All concentrations of the positive control were tested in triplicate.
- Preparation of the solvent, vehicle and negative controls : The solvent control for the test article and the positive control was DMSO diluted to 1% in 1% DMEM. Each plate included a set of six solvent control wells. On each plate blank wells were tested (no cells and no treatment).
- Stable dispersion obtained: Yes. No precipitates observed, per se. The test item was determined to be fully soluble in the primary solvent, DMSO, and was serially diluted to prepare the twelve x100 stock dilutions. Upon transfer of the x100 DMSO stock dilutions into 1% DMEM to prepare the x4 stock dilutions, the five to six highest concentrations were observed to be cloudy. Microscopic observations of the treated cultures at the end of the 48-hour treatment showed oily droplets in the top three to four doses. In first definitive test (B1), the cultures were obscured from observation at the highest dose of 2000 μM, and normal cellular morphology was documented at lower doses. In the second definitive test (B2), the cultures were obscured from observation at the two highest doses, and low levels of toxicity were documented at doses ranging from 62.5 to 500 μM. In the third definitive test (B3), the cultures showed low levels of toxicity at doses ranging from 31.3 to 125 μM, moderate levels of toxicity at doses ranging from 250 to 1000 μM, and a high level of toxicity at the 2000 μM dose. The MTT viability assay results from trials B1 and B2 generally showed viability responses that reflected the low to non-cytotoxic responses observed microscopically, while the MTT viability assay results of trial B3 showed increasing levels of MTT reduction with increases in test article concentration. The variability in test results among the three trials likely reflects variability in bioavailability of the partially soluble test article, and the increasing levels of MTT reduction reflect the test article’s ability to directly reduce MTT. Since the viability values were typically above 70% at all doses, no IC30 or IC50 values were determined. Furthermore, the test item induced a dose-related increase in luciferase gene induction, with statistically significant gene induction above the 1.5-fold threshold at several doses where viability was greater than 70%, and below the dose ranges showing solubility issues. In all three trials EC1.5 values occurred at dose well below the 2000 μM concentration, thus meeting the criteria for a positive prediction. It was therefore considered that test item solubility had limited impact on the study conclusions.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 μM (the highest test concentration required in the current guideline OECD 442C).
- Solubility in solvents: see ‘Preparation of test solutions’, ‘-Other’ for details on the solubility test and/or ‘- Stable dispersion obtained’ for further comments.
- Solubility in incubation medium: Soluble up to maximum dose level. See ‘- Stable dispersion obtained’ for further comments.
- Cytotoxicity assessment performed: Yes. See ‘- Stable dispersion obtained’ above for further comments.
- Final concentration range selected on basis of: absence of cytotoxicity (i.e. based upon % viability) of the test item. The concentration ranges were in accordance with the OECD TG 442D guideline including testing up to the highest dose required in the current guideline (2000 μM).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplicate (3) [Denoted B1, B2 and B3, respectively].
- Number of repetitions: Minimum of Two (2). Guideline specified three (3) with third only where applicable.
- Test chemical concentrations: Final test concentrations of: Experiment B1, B2 and B3: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM (final concentration DMSO of 1% i.e. DMSO diluted to 1% in 1% DMEM).
- Application procedure: See ‘cell culture and exposure’, below.
- Exposure time: 48 hours ± 1 h.
- Study evaluation and decision criteria used: See ‘Evaluation criteria’, below. In accordance with OECD 442D.
- Description on study acceptance criteria: See ‘Acceptability criteria’, below.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): See ‘cell culture and exposure’, below.
- Incubation conditions: See ‘cell culture and exposure’, below.
- Washing conditions: See ‘cell culture and exposure’, below.
- Precipitation noted: No precipitate was observed at any dose level tested. See ‘-Other’ for details on the solubility test and/or ‘- Stable dispersion obtained’ for further comments.

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: Luciferase activity: SpectraMax® luminometer and/or MTT viability measurement: VersaMax® Plate Reader. The test system is fully validated. The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. For further information contact the test facility.
- Plate used: See ‘cell culture and exposure’, below.
- Lysate preparation:
(i) Luciferase Endpoint Assay: After 48 ± 1 hours of exposure, each white-walled culture plate was removed from the incubator and allowed to cool to room temperature for at least 30 minutes. Once at room temperature, the treatment medium were decanted from each plate. The cultures were rinsed with 250 μL of CMF-OPBS (room temperature), and the CMF-DPBS rinsate was decanted from the wells. Excess CMF-DPBS was removed by blotting the plates gently onto paper towels. Fifty microliters (50 μL) of CMF-DPBS was added to each well. Fifty microliters (50 μl) of Promega ONE-Glo'" Reagent was added to each well and the plates were allowed to incubated at room temperature in the dark for 5 to 45 minutes before being read by the Molecular Devices SpectraMax luminometer. The plates are kept away from sunlight or bright fluorescent light before reading. Bright light may cause plate phosphorescence resulting in higher background levels. Plate phosphorescence has a half-life of several minutes. The plates were read within S to 45 minutes of addition of the One-Gia'"' reagent. Each plate are placed onto the plate tray in the SpectraMax luminometer, and the luminescence determination initiated by the software. The light intensity in each well is measured at 570 nm in the form of relative light units (RLUs), with dark adapt for at least 1 second and integrate activity in each well for 2 seconds.
(ii) MTT viability Cytotoxicity Assay: One of two methods were used (a) or (b)
(a) A 0.59 mg/ml MTT solution was prepared in 1% DMEM and used within 2 hours. After 48 ± 1 hours, the clear 96-well plates designated for the MTT endpoint was removed from the incubator. The test item dilutions were decanted from the plates. No was performed. Two hundred microliters (200 μL) of 1% DMEM containing 0.59 mg/ml MTT were added to each well. The plate were then incubated with a plate seal at standard culture conditions for 4 ± 0.3 hrs. Subsequently, the MTT solution was decanted, the plate blotted and 200 μl of 10% SLS added to each well. The plates were covered with a plate seal and incubated at standard culture conditions overnight. After the overnight incubation, the plate(s) are placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (ODs10) of each well was measured with the VersaMax plate reader with shaking function selected. Relative survival is obtained by comparing the amount of MTT reduction by test item treated groups to the MTT reduction by the solvent treated group on the same plate. Alternatively:
(b) A 1 mg/ml MTT solution is prepared in 1% DMEM and used within 2 hours. After 48 ± 1 hours, the clear 96-well plates designated for the MTT endpoint are removed from the incubator. The test item dilutions are decanted from the plates. No rinsing is performed. Two hundred microliters (200 μl) of 1% DMEM containing 1 mg/ml MTT was added to each well. The plate were incubated with a plate seal at standard culture conditions for 3 ± 0.2 hours. After 3 ± 0.2 hours, the MTT solution was decanted, the plate were blotted, and 200 μL of isopropanol are added to each well. The plate is covered with a plate seal, and either stored in the refrigerator (2 to 8°C) overnight prior to shaking, or immediately placed on a plate shaker and shaken for at least 30 minutes at room temperature. The absorbance at 570 nm (OD!i70) of each well is measured with the VersaMax plate reader with the shaking function selected. Relative survival is obtained by comparing the amount of MTT reduction by test item treated groups to the MTT reduction by the solvent treated group on the same plate.
- Other: Not applicable.

DATA EVALUATION
- Cytotoxicity assessment: See ‘Cell Viability Assay MTT’, below and/or previous text.
- Prediction model used: See ‘Evaluation criteria’, below. In accordance with OECD 442D.
- Other: Not applicable.

- ACCEPTABILITY CRITERIA:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, should be above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1 (B1): 4.56 µM; Experiment 2 (B2): < 4.00 µM; Experiment 3 (B3): < 4.00 (i.e. all were >1.5 threshold at up to 64 µM)
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Cinnamic aldehyde at 64 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 4.56 µM and < 4.00 µM in experiment 1 and 2 plus 3, respectively and the dose response in both experiments was greater than 2-fold (Imax was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively).
(iii) average coefficient of variation (CoV) and/or the standard deviation (SD) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: standard deviation varied between 10.56% to 16.08%

CELL LINE USED
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. The cell line was developed by the supplier (full details in the full study report). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium. The full cell culture procedures and routine passaging procedure (every 2 to 4 days) is documented in the full study report.

CELL CULTURE AND EXPOSURE
(i) When the cell culture flask(s) are 60 to 90% confluent, the KeratinoSens cells are prepared for seeding in the 96-wcll plates. The medium is aspirated, and the cell sheet rinsed twice with ca. 10 mL of DPBS containing 0.05% EDTA for approximately 2 minutes. One millilitre (1 mL) of trypsin/EDTA is added to each flask to cover the cell sheet. The flask(s) are then incubated at standard culture conditions for 5 to 10 minutes (or until they become dislodged). When more than 50% of the cells become dislodged, the flask(s) are rapped sharply against the palm of the hand. When more than 90% of the cells become dislodged, the cells are re-suspended in 5 ml Assay Medium (without G418) per flask. The concentration of cells is determined with a Coulter counter or hemocytometer. Cells are diluted to approximately 1.0 x 10^5 cells/mL in Assay Medium.
(ii) Each definitive test has 4 plates; 3 white-walled plates are used for the luciferase endpoint, while 1 clear bottomed plate is used for the cytotoxicity endpoint. These plates are designated as the x1 plates. Each 96-well plate is uniquely numbered and labelled with the seeding density, cell type, and date of seeding. Cell suspensions of 1 x10^5 cells/mL In the Assay Medium is prepared. One hundred microliters (100 μL) of the cell suspension is added to the designated wells on the 96-well plate {i.e. all wells except well H12, which is left blank). For example, using 8 tips, 100 μL of the cell suspension is placed into the first 11 columns of the 96-well tissue culture microtiter plate; one tip are removed and 100 μL is added to the remaining column of wells (Al2 -Gl2) (1 x10^5 cells/mL = 10,000 cells/well). Using a pipette, 100 μL of Assay Medium is placed into the well designated as blank {H12}. The cells are incubated for 24 ± 1 hours at standard culture conditions.
(iii) At least 2 valid definitive trials are performed for each test item. Each plate consists of up to 12 concentrations of up to 7 test article(s), a solvent control (6 wells), a series of 5 concentrations of the positive control, and a single blank well . Each test item concentration is tested in a single well. Each positive control concentration is tested in a single well. On the day following seeding, visual observations are performed on the clear plate{s) for appropriate cell growth, uniform seeding, and confluence. Prior to treatment, the Assay Medium is removed from the culture plates by careful inversion and blotting of the plates onto sterile paper towels. The cells are re-fed with 150 μL of pre-warmed (37°C) 1% DMEM (DMEM containing 1% Fetal Bovine Serum). After re-feeding, the plates are incubated at standard culture conditions until treatment.
(iii) The x10 DMSO Master Plate(s) are labelled with the appropriate test item and control designations and prepared according to the standard plate map.
(a) For the solvent control, 150 μL of DMSO is added to wells H1-H6 ; (b) for the positive control, 150 μL of DMSO is added to wells H7-H10 and/or; (c) For test item(s) diluted in DMSO, 150 μL of DMSO is added to each well in the relevant rows, except for the well(s} in column 12; for test item(s) diluted in DiH2O, 150 μL of DiH2O are added to each well in the relevant rows, except for the well(s) In column 12. The serial dilutions of the test item and the positive control are prepared in the 96-well plate. The highest stock dilutions of the test item(s) and positive control are prepared according to their dilution schemes and then added to the appropriate wells on the 96-well plate.
Subsequently, ca. 300 μL of each test article(s) highest stock dilution are added to the designated well in column 12 of the x100 DMSO Master Plate and/or ca. 300 μL of the positive control's highest stock dilution are added to well H11 on the x100 DMSO Master Plate.
The highest stock concentrations for the test item(s) and the positive control are then serially diluted in the DMSO Master Plate to create the other 11 test item, or 4 positive control dilutions. Starting with the highest stock dilution, 150 μL of each concentration is removed from the higher concentration dilution and added to the adjacent well to the left and mixed. This process of removing 150 μL from the higher dilution and adding it to the adjacent well (to the left} is repeated until the lowest concentrations is achieved.
Other information on media utilised:
(i) Assay Medium: Dulbecco's Modified Eagle Medium (DMEM) containing GlutaMAX I, 1000 mg/L D-Glucose, Sodium Pyruvate (Gibco) supplemented to contain 9.1% fetal bovine serum (FBS)
(ii) Maintenance Medium: Dulbecco's Modified Eagle Medium (DMEM) containing GlutaMAX I, 1000 mg/l D-Glucose, Sodium Pyruvate supplemented to contain 9.1% FBS and 500 μg/mL Geneticin (G418)
(iii) 1% DMEM: Dulbecco's Modified Eagle Medium containing GlutaMAX I, 1000 mg/L D -Glucose, Sodium Pyruvate supplemented to contain 1% FBS
(iv) Dulbecco's Phosphate Buffered Saline (DPBS) without Calcium & Magnesium supplemented to contain 0.05% ethylenediaminetetraacetic acid (EDTA)

The following parameters are calculated in the KeratinoSens test method:
(i) The maximal average fold induction of luciferase activity (Imax) value observed at any concentration (CImax) of the tested chemical and positive control
(ii) 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) are obtained
(iii) The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

- CELL VIABILITY ASSAY MTT
Test item IC50: IC50 value as the concentration in μM reducing the viability by 50%
Experiment 1 (B1): mean (n=3) > 2000 ; Experiment 2 (B2): mean (n=3) > 2000 ; Experiment 3 (B3): mean (n=3) > 2000
Test item IC30: IC30 value as the concentration in μM reducing the viability by 30%
Experiment 1 (B1): mean (n=3) > 2000 ; Experiment 2 (B2): mean (n=3) > 2000 ; Experiment 3 (B3): mean (n=3) > 2000

Conclusion:
The test item showed no cytotoxicity (< 70% viability). The viability of the cells was higher than 70% within the concentration ranges both experiments and IC30 and IC50 values were therefore not calculated. No precipitate was observed at any dose level tested. Specifically: the test item was determined to be fully soluble in the primary solvent, DMSO, and was serially diluted to prepare the twelve x100 stock dilutions. Upon transfer of the x100 DMSO stock dilutions into 1% DMEM to prepare the x4 stock dilutions, the five to six highest concentrations were observed to be cloudy. Microscopic observations of the treated cultures at the end of the 48-hour treatment showed oily droplets in the top three to four doses. In first definitive test (B1), the cultures were obscured from observation at the highest dose of 2000 μM, and normal cellular morphology was documented at lower doses. In the second definitive test (B2), the cultures were obscured from observation at the two highest doses, and low levels of toxicity were documented at doses ranging from 62.5 to 500 μM. In the third definitive test (B3), the cultures showed low levels of toxicity at doses ranging from 31.3 to 125 μM, moderate levels of toxicity at doses ranging from 250 to 1000 μM, and a high level of toxicity at the 2000 μM dose. The MTT viability assay results from trials B1 and B2 generally showed viability responses that reflected the low to non-cytotoxic responses observed microscopically, while the MTT viability assay results of trial B3 showed increasing levels of MTT reduction with increases in test article concentration. The variability in test results among the three trials likely reflects variability in bioavailability of the partially soluble test article, and the increasing levels of MTT reduction reflect the test article’s ability to directly reduce MTT. Since the viability values were typically above 70% at all doses, no IC30 or IC50 values were determined. Furthermore, the test item induced a dose-related increase in luciferase gene induction, with statistically significant gene induction above the 1.5-fold threshold at several doses where viability was greater than 70%, and below the dose ranges showing solubility issues. In all three trials EC1.5 values occurred at dose well below the 2000 μM concentration, thus meeting the criteria for a positive prediction. It was therefore considered that test item solubility had limited impact on the study conclusions.

- LUCIFERASE ASSAY
Imax indicating maximum fold-induction up to concentration 2000 μM
Experiment 1 (B1): mean (n=3) 5.09 ; Experiment 2 (B2): mean (n=3) 5.06 ; Experiment 3 (B3): mean (n=3) 4.97
Since Imax was > 1.5 fold, the EC1.5 was subsequently determined.

- DETERMINATIONS
EC1.5
Experiment 1: Dose related luminescence activity induction was observed after treatment with test item : EC1.5 = 4.49 μM
Experiment 2: Dose related luminescence activity induction was observed after treatment with test item : EC1.5 = 6.55 μM
Experiment 3: Dose related luminescence activity induction was observed after treatment with test item : EC1.5 = 3.20 μM

EVALUATION CRITERIA
Test item considered ‘negative’ where
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (e.g. 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
Negative results obtained with concentrations <1000 µM or 200 µg/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

RESULTS
3 out of 3 positive experiments (each in triplicate). The cells were in these experiments incubated with the test item in a concentration range of 0.977 – 2000 µM (2-fold dilution steps) for 48 hours ± 1 h. 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 test item showed no (cyto)toxicity (IC30 and IC50 values were > 2000 μM in experiment 1, 2 and 3, respectively). A biologically relevant induction of the luciferase activity was measured , with EC1.5 values of 4.49 μM, 6.55 μM and/or 3.20 μM in experiments 1, 2 and 3 respectively. The maximum luciferase activity induction (Imax) was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively. The test item is classified as positive in the KeratinoSens assay since positive results (> 1.5-fold induction) were observed at test concentrations up to 2000 μM.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, should be above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1 (B1): 4.56 µM; Experiment 2 (B2): < 4.00 µM; Experiment 3 (B3): < 4.00 (i.e. all were >1.5 threshold at up to 64 µM)
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Cinnamic aldehyde at 64 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 4.56 µM and < 4.00 µM in experiment 1 and 2 plus 3, respectively and the dose response in both experiments was greater than 2-fold (Imax was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively).
(iii) average coefficient of variation (CoV) and/or the standard deviation (SD) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: standard deviation varied between 10.56% to 16.08%

Key result

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Remarks:

/ µM

Value:

4.49 µM

Cell viability:

IC50 value as the concentration in μM reducing the viability by 50% was > 2000 μM
IC30 value as the concentration in μM reducing the viability by 30% was > 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Remarks:

/ µM

Value:

6.55 µM

Cell viability:

IC50 value as the concentration in μM reducing the viability by 50% was > 2000 μM
IC30 value as the concentration in μM reducing the viability by 30% was > 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Remarks:

/ µM

Value:

3.2 µM

Cell viability:

IC50 value as the concentration in μM reducing the viability by 50% was > 2000 μM
IC30 value as the concentration in μM reducing the viability by 30% was > 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. For further information contact the test facility.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, should be above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1 (B1): 4.56 µM; Experiment 2 (B2): < 4.00 µM; Experiment 3 (B3): < 4.00 (i.e. all were >1.5 threshold at up to 64 µM)
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Cinnamic aldehyde at 64 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 4.56 µM and < 4.00 µM in experiment 1 and 2 plus 3, respectively and the dose response in both experiments was greater than 2-fold (Imax was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively).
(iii) average coefficient of variation (CoV) and/or the standard deviation (SD) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: standard deviation varied between 10.56% to 16.08%.

Interpretation of results:

other: The test item gave 3 out of 3 positive experiments (each in triplicate). The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

Under the condition of this study, the test item is considered to be potentially sensitising to the skin. The test item gave 3 out of 3 positive experiments (each in triplicate).The test item is classified as positive in the KeratinoSens assay since positive results (> 1.5-fold induction) were observed at test concentrations up to 2000 μM.

Executive summary:

The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in at least two independent experiments. A solubility test was performed. The test item was suspended in DMSO at the highest x100 concentration of 2000 μM. The test item was found to be soluble in DMSO and formed a ‘clear colourless (non-viscous) solution’. DMSO was then utilised to further dilute test items within the definitive trials. No precipitation was reported during solubility trials. Prior to definitive testing, The ability of the test item to directly reduce MTT was assessed. Approximately 100 μL of the highest concentration of the x100 test item dilutions in DMSO were added to 1 mL of a 1.0 mg/mL MTT solution and then incubated in the dark at standard culture conditions (37 ± 1 °C humidified air containing 5 ± 1% CO2) for one hour. 100 μL of the DMSO solvent control in 1 mL of a 1.0 mg/mL MTT solution was tested concurrently. If the MTT solution colour turned blue/purple, the test article was presumed to have reduced the MTT. Within the study, the test item was observed to directly reduce MTT. In the main experiments the test item was dissolved in DMSO at the highest x100 concentration of 2000 μM. From this the test item solution was diluted to final x1 concentrations in 1% DMEM exposure medium. Resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM. The highest test concentration was the highest dose required in the current guideline.

The test item showed no (cyto)toxicity (IC30 and IC50 values were > 2000 μM in experiment 1, 2 and 3, respectively). A biologically relevant induction of the luciferase activity was measured , with EC1.5 values of 4.49 μM, 6.55 μM and/or 3.20 μM in experiments 1, 2 and 3 respectively. The maximum luciferase activity induction (Imax) was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively. The test item is classified as positive in the KeratinoSens assay since positive results (> 1.5-fold induction) were observed at test concentrations up to 2000 μM. Within the definitive tests, it was noted upon transfer of the x100 DMSO stock dilutions into 1% DMEM to prepare the x4 stock dilutions, the five to six highest concentrations were observed to be cloudy. Microscopic observations of the treated cultures at the end of the 48-hour treatment showed oily droplets in the top three to four doses. In first definitive test (B1), the cultures were obscured from observation at the highest dose of 2000 μM, and normal cellular morphology was documented at lower doses. In the second definitive test (B2), the cultures were obscured from observation at the two highest doses, and low levels of toxicity were documented at doses ranging from 62.5 to 500 μM. In the third definitive test (B3), the cultures showed low levels of toxicity at doses ranging from 31.3 to 125 μM, moderate levels of toxicity at doses ranging from 250 to 1000 μM, and a high level of toxicity at the 2000 μM dose. The MTT viability assay results from trials B1 and B2 generally showed viability responses that reflected the low to non-cytotoxic responses observed microscopically, while the MTT viability assay results of trial B3 showed increasing levels of MTT reduction with increases in test article concentration. A minor deviation to test guideline has to be noted in B3 test where the MTT viability incubation was for 22 minutes rather than 4 ± 0.3 hrs. This was not found to have adversely affected the study. The variability in test results among the three trials likely reflects variability in bioavailability of the partially soluble test article, and the increasing levels of MTT reduction reflect the test article’s ability to directly reduce MTT. Since the viability values were typically above 70% at all doses, no IC30 or IC50 values were determined. The test item induced a dose-related increase in luciferase gene induction, with statistically significant gene induction above the 1.5-fold threshold at several doses where viability was greater than 70%, and below the dose ranges showing solubility issues. In all three tests the EC1.5 values occurred at dose well below the 2000 μM concentration, thus meeting the criteria for a positive prediction. It was considered test item solubility had limited impact on the study conclusions. The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, should be above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1 (B1): 4.56 µM; Experiment 2 (B2): < 4.00 µM; Experiment 3 (B3): < 4.00 (i.e. all were >1.5 threshold at up to 64 µM). The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Cinnamic aldehyde at 64 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 4.56 µM and < 4.00 µM in experiment 1 and 2 plus 3, respectively and the dose response in both experiments was greater than 2-fold. The Imax was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively. The average coefficient of variation (CoV) and/or the standard deviation (SD) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls. The actual: standard deviation varied between 10.56% to 16.08%. All acceptability criteria were considered to be met. Under the conditions of this study, the test item is considered to yield a positive prediction in the KeratinoSens assay for skin sensitisation potential.

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2021 : The study was performed to the OECD TG 442C in chemico Direct Peptide Reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. The acceptance criteria were as follows: (i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9995 and SPLC r2 = 0.9999). (ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 60.85% ± 0.26% and SPCL 23.86% ± 2.81%) and/or (iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.26% and SPCL PC : SD = 2.81%). It was noted within the study, for Lysine peptide-positive control data of the peptide depletion of: 23.86%, which is below the lower bound threshold of 40.2%. Whereas the standard deviation for three replicates of positive control cinnamic aldehyde was 2.81, which is well within the acceptable range of <11.6. All the acceptance criteria for Reference control A, B and C and standard curve R2 value were met. It was considered, within the study that underprediction of positive control cinnamic aldehyde % Lysine peptide depletion should not impact the sensitisation potential prediction of the test item because regardless of Lysine data, as the test item is predicted to be potential non-sensitiser based on mean Cysteine only prediction model. (iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. The means of Reference Control samples A and C were not both within the acceptance criteria of 0.50 ± 0.05 mM. Specifically, 1) the mean Cysteine peptide concentration of the Reference Control C (Acetonitrile) was 0.4497 mM and Reference Control C (Water) was 0.4440 mM, which is outside of the range of 0.45 to 0.55 mM. It was considered within the study that this was an acceptable deviation based on the totality of information. Specifically, the peak area Coefficient of Variation (CV) was <5% for both Reference Control C-Acetonitrile and -Water, which is well within the acceptable range of <15%. Reference Control C is used in the calculation to determine percent peptide depletion of the test article mixed with peptide. Additionally, all the acceptance criteria for Reference control A and B and standard curve R2 value were met. Considering this information in totality, marginally lower mean Cysteine peptide concentration. It was considered this would have a negligible impact on sensitisation prediction in Cysteine reactivity model. Furthermore, the concurrent positive control cinnamic aldehyde had a Cysteine peptide depletion of 60.85%, meeting the assay acceptance criteria. (v) the  Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.01 to 0.02% ; Lysine: Reference Controls B and C was 0.01%). In the cysteine reactivity assay the test item showed 0.58% SPCC depletion while in the lysine reactivity assay the test item showed 0.19% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.39% and as a result the test item was considered to be in the Cysteine 1:10 / Lysine 1:50 prediction model 'no or minimal reactivity' class. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2022 : The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in at least two independent experiments. A solubility test was performed. The test item was suspended in DMSO at the highest x100 concentration of 2000 μM. The test item was found to be soluble in DMSO and formed a ‘clear colourless (non-viscous) solution’. DMSO was then utilised to further dilute test items within the definitive trials. No precipitation was reported during solubility trials. Prior to definitive testing, The ability of the test item to directly reduce MTT was assessed. Approximately 100 μL of the highest concentration of the x100 test item dilutions in DMSO were added to 1 mL of a 1.0 mg/mL MTT solution and then incubated in the dark at standard culture conditions (37 ± 1 °C humidified air containing 5 ± 1% CO2) for one hour. 100 μL of the DMSO solvent control in 1 mL of a 1.0 mg/mL MTT solution was tested concurrently. If the MTT solution colour turned blue/purple, the test article was presumed to have reduced the MTT. Within the study, the test item was observed to directly reduce MTT. In the main experiments the test item was dissolved in DMSO at the highest x100 concentration of 2000 μM. From this the test item solution was diluted to final x1 concentrations in 1% DMEM exposure medium. Resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM. The highest test concentration was the highest dose required in the current guideline. The test item showed no (cyto)toxicity (IC30 and IC50 values were > 2000 μM in experiment 1, 2 and 3, respectively). A biologically relevant induction of the luciferase activity was measured , with EC1.5 values of 4.49 μM, 6.55 μM and/or 3.20 μM in experiments 1, 2 and 3 respectively. The maximum luciferase activity induction (Imax) was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively. The test item is classified as positive in the KeratinoSens assay since positive results (> 1.5-fold induction) were observed at test concentrations up to 2000 μM. Within the definitive tests, it was noted upon transfer of the x100 DMSO stock dilutions into 1% DMEM to prepare the x4 stock dilutions, the five to six highest concentrations were observed to be cloudy. Microscopic observations of the treated cultures at the end of the 48-hour treatment showed oily droplets in the top three to four doses. In first definitive test (B1), the cultures were obscured from observation at the highest dose of 2000 μM, and normal cellular morphology was documented at lower doses. In the second definitive test (B2), the cultures were obscured from observation at the two highest doses, and low levels of toxicity were documented at doses ranging from 62.5 to 500 μM. In the third definitive test (B3), the cultures showed low levels of toxicity at doses ranging from 31.3 to 125 μM, moderate levels of toxicity at doses ranging from 250 to 1000 μM, and a high level of toxicity at the 2000 μM dose. The MTT viability assay results from trials B1 and B2 generally showed viability responses that reflected the low to non-cytotoxic responses observed microscopically, while the MTT viability assay results of trial B3 showed increasing levels of MTT reduction with increases in test article concentration. The variability in test results among the three trials likely reflects variability in bioavailability of the partially soluble test article, and the increasing levels of MTT reduction reflect the test article’s ability to directly reduce MTT. Since the viability values were typically above 70% at all doses, no IC30 or IC50 values were determined. The test item induced a dose-related increase in luciferase gene induction, with statistically significant gene induction above the 1.5-fold threshold at several doses where viability was greater than 70%, and below the dose ranges showing solubility issues. In all three tests the EC1.5 values occurred at dose well below the 2000 μM concentration, thus meeting the criteria for a positive prediction. It was considered test item solubility had limited impact on the study conclusions. The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, should be above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1 (B1): 4.56 µM; Experiment 2 (B2): < 4.00 µM; Experiment 3 (B3): < 4.00 (i.e. all were >1.5 threshold at up to 64 µM). The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Cinnamic aldehyde at 64 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 4.56 µM and < 4.00 µM in experiment 1 and 2 plus 3, respectively and the dose response in both experiments was greater than 2-fold. The Imax was 46.63-fold, 62.45-fold and 62.13-fold in experiment 1, 2 and 3, respectively. The average coefficient of variation (CoV) and/or the standard deviation (SD) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls. The actual: standard deviation varied between 10.56% to 16.08%. All acceptability criteria were considered to be met. Under the conditions of this study, the test item is considered to yield a positive prediction in the KeratinoSens assay for skin sensitisation potential.

 

3. Molecular initiating Key Event 3: No data.

 

4. QSAR : Skin sensitization GHS v.02.04, OASIS TIMES v2.31.2, 2022 :
OASIS TIMES v2.31.2, Skin sensitization GHS v.02.04
Results:
The parent chemical is a mixture or multi-constituent substance
Represented by generic PARENT SMILES : prediction: GHS Skin Sensitiser 1B
Conclusion: The substance is expected to be a skin sensitiser. Based on in silico battery screening: there is potential for formation of (n=1) : GHS Skin Sensitiser 1B metabolite. No other metabolites are predicted to be sensitisers. Consequentially, based on weight of evidence and/or based on precautionary principle: substance is predicted to be GHS Skin Sensitiser sub-category 1B.
(ACF: 50% correct, 0% incorrect, 50% unknown)

 

Weight of Evidence Conclusion:
The applicant assesses by expert judgement the available information. Within the battery of in silico modelling and in vitro test assays, there is are negative (or equivocal) to positive predictions for sensitisation. The test item appears to be either a: non-sensitiser to weak sensitiser. The mechanistic basis is predicted to be based upon metabolism by (Q)SAR screening which would explain KERATINOSENS assay (OECD TG 442D) prediction due to some metabolic competency (see references). Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. The substance is assigned to GHS Classification: Skin Sensitisation Category 1B.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation: category 1B.

Within the battery of in silico modelling and in vitro test assays, there is are negative (or equivocal) to positive predictions for sensitisation. The test item appears to be either a: non-sensitiser to weak sensitiser. Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. The substance is assigned to GHS Classification: Skin Sensitisation Category 1B.

 

References:
(1) ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, July 2017)
(2) OECD (2017) Guidance Document on the Reporting of Defined Approaches and Individual Information Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitisation. OECD Series on Testing and Assessment No. 256
(3) OECD TG 497, June 2021
(4) OECD 336 (2021) : Supporting Document to the OECD Guideline 497 On Defined Approaches For Skin Sensitisation
(5) OECD (2018) : Supporting document for evaluation and review of draft Guideline (GL) for Defined Approaches (DAs) for Skin Sensitisation