2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Endpoint summary

Description of key information

DPRA CRL 2020

The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Keratinosens CRL 2020

The test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

DEREK CRL 2020

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

WoE of in vitro data CRL 2020

Based on the results, it is concluded that the test item is a skin sensitizer. In the absence of potency data for the test item it is recommended to perform an in vivo test to determine the skin sensitizing potential of the test item and classify accordingly.

In vivo LLNA CRL 2020

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%. Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

23 November 2019 to 06 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

2. MODEL (incl. version number)
DEREK NEXUS version 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C1=CC=CC=C1C=2C=CC(=CC=2)C(C3=C(C=CC=C3)C(OCC(CCCC)CC)=O)=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain There is proof that the proposition is true.
Probable There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible The weight of evidence supports the proposition.
Equivocal There is an equal weight of evidence for and against the proposition.

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

DEREK NEXUS contains an expert-derived functionality that can provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of skin sensitization data, producing the following outcomes:
- In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
- For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified1. features are highlighted to enable the negative prediction to be verified by expert assessment.
- In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified2. features are highlighted to enable the negative prediction to be verified by expert assessment.

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:
1Misclassified features are those that have been derived from non-alerting substances in the Lhasa test reference set
2Unclassified features are those that have not been found in the Lhasa test reference set

EC3Q = MWQ /(Σ ωNN / Σ TNN)

MW = molecular weight
T = Tanimoto similarity score
ω = weighting factor = (MWNN/EC3) * TNN
Q = query compound
NN = nearest neighbour

The EC3 is the estimated concentration needed to produce a stimulation index of 3.

5. APPLICABILITY DOMAIN
See QPRF/QMRF

6. ADEQUACY OF THE RESULT
See QPRF/QMRF

Qualifier:

according to guideline

Guideline:

other: DEREK NEXUS

Version / remarks:

Version 6.0.1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Molecular formula: C28H30O3
Molecular weight: 414.22 g/mol

Parameter:

other: Prediction

Remarks on result:

positive indication of skin sensitisation

The result as generated by DEREK NEXUS is presented in Appendix 2 (appended to attached background material). The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3 (appended to attached justification).

Interpretation of results:

GHS criteria not met

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

Executive summary:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 Nov 2019 - 28 Jul 2020

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))

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Molecular formula: C28H30O3
Molecular weight: 414.22

Details on the study design:

PREPARATION OF TEST ITEM
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e., by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol and ethanol.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 64.07 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1547 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

TEST SYSTEM
Test system Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Rationale Recommended test system in the international OECD guideline for DPRA studies.
Source JPT Peptide Technologies GmbH, Berlin, Germany.
Batch See Appendix 7 for detailed information.
Storage The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

REAGENTS
Acetone HiPersolv, VWR international, Amsterdam, The Netherlands
Acetonitrile (ACN) HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate BioSolve, VWR international
Ammonium hydroxide 25%, Merck, Darmstadt, Germany
Dimethylsulfoxide (DMSO) Seccosolv, Merck
Disodium hydrogen phosphate(Na2HPO4·12H2O) Emsure, Merck
Ethanol LiChrosolv, Merck
Isopropanol (IPA) LiChrosolv, Merck
Methanol BioSolve, VWR international
Milli-Q water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate(NaH2PO4·H2O) Lab Honeywell, Seelze, Germany
Trifluoroacetic acid (TFA) >99%, Sigma Aldrich, Zwijndrecht, The Netherlands


POSITIVE CONTROL
Identification: Cinnamic aldehyde
Test Facility Test Item Number: RS473/C
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKCB9907
Purity: 99.1%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2021 (expiry date)
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Purity/composition correction factor: No correction factor required



EXPERIMENTAL DESIGN
Preparation of Solutions for Cysteine Reactivity Assay
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 11.8 mg of SPCC in 23.55 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

SPCC Calibration Curve
A SPCC calibration curve was prepared as described in table 3 below.

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in table 4 below.

Preparation of Solutions for Lysine Reactivity Assay
Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.6 mg of SPCL in 20.46 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in table 5 below.

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in table 6 below.

Sample Incubations
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 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.8 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:

System 1 (used for Cysteine Reactivity Assay):
- Alliance separations module 2695 (Waters, Milford, MA, USA)
- Dual λ absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
- Alliance separations module 2695 (Waters, Milford, MA, USA)
- Dual λ absorbance detector 2487 (Waters)

All samples were analyzed according to the HPLC method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have a r2>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.


ANALYSIS
Data Evaluation
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 according to the following formula:

Percent Peptide Depletion= [1 - ((Peptide Peak Area in Replicate Injection (at 220 nm)) / (Mean Peptide Peak Area in Reference Controls (at 220 nm)))] × 100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
Data Interpretation
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table 7 below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed in table 8 below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Run / experiment:

other: SPCC Depletion

Parameter:

other: Cysteine reactivity

Value:

9.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: SPCL Depletion

Parameter:

other: Lysine reactivity

Value:

34.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Mean of SPCC and SPCL depletion

Parameter:

other: DPRA Prediction and Reactivity Classification

Value:

22.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Solubility Assessment of the Test Item

At a concentration of 100 mM, C991 was not soluble in MQ, ACN:MQ (1:1, v/v) and methanol, but was soluble in ACN, IPA, acetone:ACN (1:1, v/v) DMSO:ACN (1:9, v/v) and ethanol. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study.

Cysteine Reactivity Assay

The reactivity of C991 towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC analysis, following 24.8 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-210244/A and 210244/A-cys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.521 ± 0.006 mM while the mean peptide concentration of Reference Controls C was 0.521 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.6%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6

(Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 37.51. The mean A220/A258 ratio ± 10% range was 33.76-41.26. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.2% ± 0.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM C991 stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see

chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 210244/A-cys samples, the mean SPCC A220/A258 area ratio was 37.89. Since this was within the 33.76-41.26 range, this again indicated that there was no co elution of the test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 9.8% ± 12.3%.

Lysine Reactivity Assay

The reactivity of C991 towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC analysis, following 24.8 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-210244/A and 210244/A-lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9999. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.497 ± 0.010 mM while the mean peptide concentration of Reference Controls C was 0.530 ± 0.032 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 5.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 30.71. The mean A220/A258 ratio ± 10% range was 27.64-33.78. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 66.4% ± 0.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM C991 stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item

with SPCL. For the 210244/A-lys samples, the mean SPCL A220/A258 area ratio was 30.40. Since this was within the 27.64-33.78 range, this again indicated that there was no co elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 34.3% ± 15.9%.

The standard deviation value of the SPCL depletion for the test item was 15.9% which is outside specification (i.e., acceptance criteria <11.6%). As the acceptance criteria related to the positive control and the reference control with SPCL were within specification, an instrumental issue leading to a SD of SPCL depletion for the test item >11.6% was unlikely. The SD of SPCC depletion for the test item was also high (i.e., 12.3%). These high SD’s were most likely test item related (i.e., due to the precipitation of the test item in the peptides test item samples) and the SPCL test item results were therefore accepted.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 9.8% SPCC depletion while in the lysine reactivity assay the test item showed 34.3% SPCL depletion. The mean of the SPCC and SPCL depletion was 22.1% and as a result the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Text Table 10       

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
9.80%	±12.3%	34.30%	±15.9%^1	22.10%	Positive: Low reactivity

SD = Standard Deviation.

¹ The standard deviation value of the SPCL depletion for the test item was 15.9% which is outside specification (i.e., acceptance criteria <11.6%). As the acceptance criteria related to the positive control and the reference control with SPCL were within specification, an instrumental issue leading to a SD of SPCL depletion for the test item >11.6% was unlikely. The SD of SPCC depletion for the test item was also high (i.e., 12.3%). These high SD’s were most likely test item related (i.e., due to the precipitation of the test item in the peptides test item samples) and the SPCL test item results were therefore accepted.

Interpretation of results:

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

Conclusions:

In conclusion, this DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of the test item towards 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 spectrophotometric detection at 220 nm and 258 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.

The study procedures described in this report were based on the most recent OECD guideline.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Text Table 1       Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.998	>0.99	0.9999
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.521 ± 0.006	0.50 ± 0.05	0.497 ± 0.010
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.521 ± 0.004	0.50 ± 0.05	0.530 ± 0.032
CV (%) for RC samples	<15.0	1.6	<15.0	5.4
B and C
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	74.2	40.2-69.0	66.4
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.7
SD of peptide depletion for the test item (%)	<14.9	12.3	<11.6	15.9

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the SPCC depletion for the test item, were all within the acceptability criteria for the DPRA.

The standard deviation value of the SPCL depletion for the test item was 15.9% which is outside specification (i.e., acceptance criteria <11.6%). As the acceptance criteria related to the positive control and the reference control with SPCL were within specification, an instrumental issue leading to a SD of SPCL depletion for the test item >11.6% was unlikely. The SD of SPCC depletion for the test item was also high (i.e., 12.3%). These high SD’s were most likely test item related (i.e., due to the precipitation of the test item in the peptides test item samples) and the SPCL test item results were therefore accepted.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 9.8% SPCC depletion while in the lysine reactivity assay

the test item showed 34.3% SPCL depletion. The mean of the SPCC and SPCL depletion was 22.1% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Text Table 2       SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
C991	9.80%	±12.3%	34.30%	±15.9%^1	22.10%	Positive: Low reactivity

SD = Standard Deviation.

1 The standard deviation value of the SPCL depletion for the test item was 15.9% which is outside specification (i.e., acceptance criteria <11.6%). As the acceptance criteria related to the positive control and the reference control with SPCL were within specification, an instrumental issue leading to a SD of SPCL depletion for the test item >11.6% was unlikely. The SD of SPCC depletion for the test item was also high (i.e., 12.3%). These high SD’s were most likely test item related (i.e., due to the precipitation of the test item in the peptides test item samples) and the SPCL test item results were therefore accepted.

In conclusion, this DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 November 2019 - 04 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

March, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Molecular formula: C28H30O3
Molecular weight: 414.22


Solubility in vehicle: DMSO: Not indicated

Stability in vehicle: DMSO: Not indicated

Details on the study design:

DOSE FORMULATION
Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (Clear Colourless). The 100-fold dilution of the 200, 100, 50, 25, 13 and 6.3 mM DMSO stock formed a homogeneous solution (slight precipitation). The 100-fold dilution of the 3.1 and 1.6 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation). The 2000 µM concentration (200 mM stock) was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test item was dissolved in DMSO at 200 mM (Clear Colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
Precipitation was observed at the start and end of the incubation period at test concentrations of 250 µM and upwards in the 96-well plates in both experiments.
Test item concentrations were used within 3 hours after preparation.
Any residual volumes were discarded.

Preparation of the Positive Control
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.

Preparation of the Vehicle Control
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

Blank
On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM
Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). 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.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

CELL CULTURE
Basic medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

Exposure medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 57 – 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

EXPERIMENTAL DESIGN
Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+7 in experiment 1 and P+9 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C
± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Positive control results:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

2.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Remarks:

(µM)

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

2.89

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Remarks:

(µM)

Value:

30

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 test item was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table 1 (Appendix 1) and Figure 2-3 (Appendix 2). The results of the positive control are summarized in Table 2 (Appendix 1) and Figure 4-5 (Appendix 2). An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3. The individual raw data are presented in Appendix 3 and Appendix 4. The historical control data are presented in Appendix 5.
Appendices 1 - 5 are appended to the background 'attached background material'.

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 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.

Experiment 1
- Precipitation was observed at the start and end of the incubation period in the 96-well plates at test concentrations of 250 µM and upwards.
- The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.54 and the EC1.5 2.6 µM.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.91 and the EC1.5 30 µM.

Experiment 2
- Precipitation was observed at the start and end of the incubation period in the 96-well plates at test concentrations of 250 µM and upwards.
- The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.89 and the EC1.5 0.81 µM.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.84 and the EC1.5 24 µM.

Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (30 µM and 24 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.91-fold and 3.84-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 8.8% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
The test item showed no toxicity (no IC30 and IC50 value) and a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 2.6 µM and 0.81 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.54-fold and 2.89-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

Interpretation of results:

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

Conclusions:

In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

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^TM assay.

The study procedures described in this report were based on the most recent OECD guideline.

The test item was a colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in est concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item precipitated at test concentrations of 250 µM and upwards. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.  

- The EC1.5 of the positive control was within two standard deviations of the historical mean (30 µM and 24 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.91-fold and 3.84-fold in experiment 1 and 2, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 8.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item showed no toxicity (no IC30 and IC50 value) and a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 2.6 µM and 0.81 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.54-fold and 2.89-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 4

Endpoint:

skin sensitisation: in vitro

Remarks:

WoE report

Type of information:

other: Weight of Evidence

Adequacy of study:

weight of evidence

Study period:

06 August 2020

Reliability:

other: Weight of Evidence

Qualifier:

no guideline required

Principles of method if other than guideline:

The objective of this study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico, in chemico and in vitro data.
A DEREK assessment, a DPRA and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Weight of Evidence

Specific details on test material used for the study:

CAS number 75005-95-7
EC number 278-051-5
Chemical Name 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
Molecular weight 414.22 g/mol
Purity/Composition 96.27%
UVCB No

Details on the study design:

BACKGROUND/SCOPE
The weight of evidence approach is based on in silico, in chemico and in vitro data, addressing each of the following key events of skin sensitization on its own or together:
1. Key event 1: Covalent binding of the electrophilic substance to proteins; tested by OECD 442C: Direct Peptide Reactivity Assay (DPRA)
2. Key event 2: Release of pro-inflammatory cytokines and induction of cyto-protective pathways in keratinocytes; tested by OECD 442D: ARE-Nrf2 Luciferase Test Method or KeratinoSensTM assay
3. Key event 3: Activation and maturation of dendritic cells; OECD 442E Myeloid U937 Skin Sensitization Test (U-SENSTM)
4. Key event 4: Presentation of the chemical allergen by dendritic cells to naïve T-cells, which leads to their differentiation and proliferation into allergen-specific memory T-cells; no generally accepted in vitro test available yet.
If information from test method(s) addressing one or two of these key events allows classification and risk assessment according to point 8.3 of Annex VII of the REACH Regulation, studies addressing the other key event(s) need not be conducted.

According to the Guidance on information requirements and chemical safety assessment R7a (v.6.0 July 2017), to reach a conclusion on (non-)classification, the following questions should be addressed:
- Does the evidence enable to conclude that the substance is not a skin sensitizer? If so, conclude on no classification.
- Does the evidence enable to conclude that the substance is presumed to produce significant sensitization in humans i.e. Cat. 1A? If so, classify accordingly.
- Does the evidence enable to conclude that the substance is a skin sensitizer and significant sensitization in humans i.e. Cat. 1A can be excluded? If so, it is presumed that the substance would be a moderate skin sensitizer i.e. Cat. 1B and it is recommended to classify accordingly.

In case none of these conditions are met, e.g. when Cat. 1A cannot be excluded, further testing needs to be performed, in vivo testing being the last resort. At the moment no accepted in vitro studies are available to discern between Cat. 1A and 1B.
In case of positive in chemico and in vitro skin sensitization tests and absence of reliable indication for potency by DEREK, for the time being performance of an in vivo study is the only option to determine the degree of potency (see CLP Regulation 3.4 Respiratory or skin sensitisation). 

METHOD
As the substance is a mono-constituent, the following strategy was followed:
STEP 0: The starting point for the weight of evidence is the assessment whether new studies are required. Available information on the test item was used to evaluate whether:
- The substance is not a strong acid (pH≤ 2.0) or base (pH ≥ 11.5), known to be not corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature.
- No adequate existing human data, which provide evidence that the substance is a skin sensitizer are available.
- No data from existing studies on skin sensitization in laboratory animals, which provide sound conclusive evidence that the substance is a sensitizer or non-sensitizer are available.
This step is the responsibility of the sponsor.

If no reliable data on solubility is available, a solubility test is performed at Charles River to determine whether the substance dissolves sufficiently in a solvent which is appropriate for each test mentioned below. In case the solubility test demonstrates solubility of the test substance that meets the precondition limits for the in vitro tests, the following step-wise testing approach is followed:

STEP 1: DEREK assessment (overall skin sensitizing events), Direct Peptide Reactivity Assay (DPRA; molecular interaction with skin proteins) and KeratinoSensTM assay (inflammatory response in keratinocytes) are performed.

STEP 2: Depending on the outcome of the studies performed in STEP 1 and in absence of a definite conclusion on possible skin sensitization, the U-SENSTM assay is performed (key event: activation of dendritic cells).
 
STEP 3: Based on a weight of evidence of all available data on the test item related to skin sensitization, an argument is prepared to conclude on the classification for the substance or, if no conclusion can be drawn, to conclude on the performance of an in vivo skin sensitization study.

Parameter:

other: Weight of evidence

Value:

0

Remarks on result:

positive indication of skin sensitisation

At the start of testing, there was no data available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment , a DPRA and a KeratinoSens^TM assay .

RESULTS OF STUDIES PERFORMED

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

A valid DPRA was performed according to OECD TG 442C and in accordance with GLP principles. The test item was dissolved in acetonitrile at 100 mM. Upon preparation as well as after incubation of the test item with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) a precipitate was observed in the co-elution controls and the test item samples. In the cysteine reactivity assay the test item showed 9.8% ±12.3% SPCC depletion. In the lysine reactivity assay the test item showed 34.3% ±15.9% SPCL depletion. The standard deviation value of the SPCL depletion for the test item was 15.9% which is outside specification (i.e., acceptance criteria <11.6%). As the acceptance criteria related to the positive control and the reference control with SPCL were within specification, an instrumental issue leading to a SD of SPCL depletion for the test item >11.6% was unlikely. The SD of SPCC depletion for the test item was also high (i.e., 12.3%). These high SD’s were most likely test item related (i.e., due to the precipitation of the test item in the peptides test item samples) and the SPCL test item results were therefore accepted. The mean value of the SPCC and SPCL depletion was 22.1% and as a result the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated

A valid KeratinoSens^TM assay was performed according to OECD TG 442D and in accordance with GLP principles. The test item was dissolved in DMSO to a final concentration of 200 mM and formed a colorless solution. Two valid independent tests were performed. The cells were incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) in both experiments. Precipitation was observed at the start and end of the incubation period at test concentrations of 250 µM and upwards. The test item showed no toxicity (no IC30 and IC50 value) and a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 2.6 µM and 0.81 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.54-fold and 2.89-fold in experiment 1 and 2, respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

DISCUSSION

The test item showed to be reactive with SPCL moieties and less with SPCC moieties. The standard deviation of the depletion values was high and is likely related to the precipitation observed in the test samples. As a result the reactivity of the test item to SPCC and SPCL moieties cannot be accurately determined however the depletion does show that interaction with nucleophilic centers can take place and it is therefore expected that the test item can interact with endogenous skin proteins. The KeratinoSens^TM assay was positive as significant luciferase induction (>1.5-fold induction) in keratinocytes was observed at low concentrations and this serves as a clear indication that keratinocytes can become activated to a low dose exposure of the test item. The DEREK assessment was negative and predicted the substance to be a non-sensitizer however considering the first two key events in the skin sensitization AOP tested positive, there are indications that the test item has skin sensitizing properties. Based on the data available, a potency assessment is not possible and a Cat. 1A classification cannot be excluded. Further testing needs to be performed to determine the potency. In the absence of accepted in vitro studies to determine the potency it is recommended to perform an in vivo study to determine the potency. The results from this test will be leading to determine whether the test item has skin sensitizing properties and to classify the substance accordingly

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results, it is concluded that the test item is a skin sensitizer. In the absence of potency data for the test item it is recommended to perform an in vivo test to determine the skin sensitizing potential of the test item and classify accordingly.

Executive summary:

The objective of this study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico, in chemico and in vitro data.

A DEREK assessment, a DPRA and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

A DEREK prediction on the skin sensitizing potential of the test item was negative. The DPRA was positive as the test item depleted SPCC and SPCL moieties. The KeratinoSensTM assay was positive as significant luciferase induction (>1.5-fold induction) was observed at low concentrations which is indicative for activation of keratinocytes when exposed to the test item. Based on two positive tests, there are indications that the test item has skin sensitizing properties. However as potency cannot be determined with the current in vitro methods it is recommended to continue with in vivo testing to determine the potency and classify the test item accordingly.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2020 - 08 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Section 4, Health Effects, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: Janvier, Le Genest-Saint-Isle, France
Females: Nulliparous and non-pregnant
Age at study initiation: Approximately 10 weeks old
Weight at study initiation: 20.0 to 28.3 g

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Diet:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Justification for Test System and Number of Animals
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in Appendix 4 of this report. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.


ENVIRONMENTAL CONDITIONS
Temperature: The actual daily mean temperature during the study period was 22 to 23°C
- Humidity: The actual daily mean relative humidity during the study period was 40 to 52%
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained


IN-LIFE DATES
Study Initiation Date: 10 Apr 2020
Initiation of Dosing: 06 May 2020
Completion of In-life: 08 Jun 2020
Experimental Start Date: 06 May 2020
Experimental Completion Date: 09 Jun 2020


Rationale for Vehicle
The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials will be retained by the Test Facility. There was no information available about the stability and solubility of the test item in vehicle.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

Vehicle:

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

Remarks:

Merck, Darmstadt, Germany and Acros Organics, Geel, Belgium

Concentration:

0%, 2%, 5%, 10%

No. of animals per dose:

5 animals per dose

Details on study design:

PRE-SCREEN TESTS:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.

Initially, two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration that could technically be applied.
Based on the results of the initially treated animals, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.

The test system, procedures and techniques were identical to those used in the main study except that the application method may have been different (see tables in Appendix 1) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

At a 100 and 50% test item concentration, clinical signs of systemic toxicity (hunched posture) were noted. In addition, at a 50% test item concentration, variation in ear thickness during the observation period was more than 25% from Day 1 pre-dose values. Therefore, these concentrations did not meet the selection criteria.
At a 25% test item concentration, signs of systemic toxicity (hunched posture) were noted. At a 10% test item concentration, no signs of systemic toxicity irritation or ear thickness above the threshold and only very slight erythema was observed. Therefore, a 10% concentration was selected as highest concentration for the main study.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Allocation - see table in 'other infromation on materials and methods' section

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Statistics:

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in June 2020, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).

The SI values calculated for the test item concentrations 5, 10 and 25% were 2.4, 2.9 and 4.5, respectively. An EC3 value of 10.9% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the Charles River Den Bosch archives. The test described above was performed in accordance with Charles River Den Bosch Standard Operating Procedures and the report was audited by the QA-unit.

Parameter:

SI

Value:

0.8

Test group / Remarks:

2% concentration

Parameter:

SI

Value:

0.8

Test group / Remarks:

5% concentration

Parameter:

SI

Value:

0.7

Test group / Remarks:

10% concentration

Parameter:

other: DPM

Value:

508

Variability:

± 138

Test group / Remarks:

Group 1

Parameter:

other: DPM

Value:

416

Variability:

± 73

Test group / Remarks:

Group 2

Parameter:

other: DPM

Value:

389

Variability:

± 27

Test group / Remarks:

Group 3

Parameter:

other: DPM

Value:

368

Variability:

± 111

Test group / Remarks:

Group 4

Cellular proliferation data / Observations:

Skin Reactions / Irritation
The very slight irritation of the ears as shown by all animals treated at 5 and 10% concentration between Days 1 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes.
Transparent test item remnants were present on the dorsal surface of the ears of all animals treated at a 5% and 10% concentration between Days 1 and 5, which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period

Macroscopic Examination of the Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 416, 389 and 368 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8, 0.8 and 0.7, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 4).
Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

The study was carried out based on the guidelines described in:

- OECD, Section 4, Health Effects, No.429 (2010).

- EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

- EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 100%, 50% and 25% test item concentration clinical signs of systemic toxicity were noted and variation in ear thickness during the observation period was more than 25% from Day 1 pre-dose values in animals dosed with 50% test item. Therefore, these concentrations did not meet the selection criteria. At a 10% test item concentration, no signs of systemic toxicity irritation or ear thickness above the threshold, and only very slight erythema was observed. Therefore, a 10% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the test item and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 416, 389 and 368 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8, 0.8 and 0.7, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The studies were conducted according to guidelines or validated QSAR both with GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is considered not to meet the criteria for skin sensitisation under the EU Classification, Lbelling, and Packaging (CLP) regulation 1272/2008.