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

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

954-543-1

Cas Number:

2489703-47-9

Molecular formula:

C20H24O2

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light
- Other: colourless (non-viscous) liquid

In chemico test system

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

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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%)

Any other information on results incl. tables

 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.

Applicant's summary and conclusion

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.