Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28-11-2018 to 04-12-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: January 2018 ; signature: June 2018

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

EC Number:

915-782-7

Molecular formula:

C12H14O

IUPAC Name:

Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, under Nitrogen, in the dark
- Other: Colourless (sponsor) ; clear colourless (laboratory)

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. This information is in the public domain.
- 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 and/or analysis. The test item achieved solubility in acetonitrile at a nominal concentration of 100 mM. Solutions of the test item in acetonitrile were successfully analysed by the validated DPRA analytical method (cited in the full study report) in both the Cysteine and Lysine containing synthetic peptides.
- HPLC-PDA (UV) methodology are reported in the full study report.
- Preparation of synthetic peptide solutions [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine and Lysine: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca. 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
2. Calibration: standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.
3. Stability control, 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).
- Sample incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were incubated, as applicable. The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.
- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = > 0.999 and SPLC r2 = > 0.999)
(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 72.6% SD: 0.15% and SPCL 59.2% SD: 0.89%)
(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 = 0.15% and SPCL PC : SD = 0.89%)
(iv) mean peptide concentration of Reference Controls is to be 0.50 ± 0.05 mM (Actual: Cysteine: reference controls: 0.506 mM CV 0.34% ; Lysine: reference controls: 0.505 mM CV 0.33%, respectively).
(v) Coefficient of Variation (CV) of peptide areas for: Reference Controls in ACN are to be <15.0%. (Actual: Reference Controls: Cysteine: CV = 0.34% ; Lysine: CV = 0.33%)
- For each peptide analysis all acceptance criteria were met: linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility
- There was no co-elution of test item with either peptide.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=751) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=776) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile

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)

Results and discussion

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 72.6% SD 0.15% (high reactivity)
- PC LYS-peptide depletion (mean): 59.2% SD 0.89% (high reactivity)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean (%)

Parameter:

other: Cys-peptide depletion

Value:

0.605

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Test item appears to have no or minimal reactivity to CYS peptide

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Results 2

Run / experiment:

other: Mean (%)

Parameter:

other: Lys-peptide depletion

Value:

-0.387

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Test item appears to have no or minimal reactivity to LYS peptide

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 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. This information is in the public domain.

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

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = > 0.999 and SPLC r2 = > 0.999)
(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 72.6% SD: 0.15% and SPCL 59.2% SD: 0.89%)
(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 = 0.15% and SPCL PC : SD = 0.89%)
(iv) mean peptide concentration of Reference Controls is to be 0.50 ± 0.05 mM (Actual: Cysteine: reference controls: 0.506 mM CV 0.34% ; Lysine: reference controls: 0.505 mM CV 0.33%, respectively).
(v) Coefficient of Variation (CV) of peptide areas for: Reference Controls in ACN are to be <15.0%. (Actual: Reference Controls: Cysteine: CV = 0.34% ; Lysine: CV = 0.33%)
- For each peptide analysis all acceptance criteria were met: linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility
- There was no co-elution of test item with either peptide.
All relevant acceptability criteria were met.

Any other information on results incl. tables

Table 1.0 – Acceptability of the DPRA

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <15%)	SD <14.9%
	Lysine	r2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r2>0.999	72.6 (SD, 0.15%, n=3)	B: 0.506 mM (CV 0.34%, n=6)	SD 0.23% (n=3)
	Lysine	r2>0.999	59.2 (SD, 0.89%, n=3)	B: 0.505 mM (CV 0.33%, n=6)	SD 0.52% (n=3)

CV: Coefficient of Variation

SD: Standard deviation

 

Table 2.0 – Results of the DPRA with the test item

	Mean peak area of peptide	Mean peak area of peptide with test item (µV.sec)	Mean peptide depletion of test samples (%)
Cysteine	Control B: 920490 (n=6)	914920 (n=3)	0.605
Lysine	Control B: 797450 (n=6)	800540 (n=3)	-0.387

Applicant's summary and conclusion

Interpretation of results:

other: The test item gave a negative and was classified in the “no to 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 Classification and Labelling purposes

Conclusions:

Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item indicated a negative in the DPRA and was classified in the “no to minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

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 synthetic peptide 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 UV 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 was found to be an appropriate solvent to dissolve the test item at up to nominal 100 mM concentration. In the cysteine reactivity assay the test item showed 0.605% SPCC depletion while in the lysine reactivity assay the test item showed -0.387% SPCL depletion. All relevant test acceptability criteria were met. Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item indicated a negative in the DPRA and was classified in the “no to minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.