4-methyl-4-phenylpentan-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 - 13 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines & Healthcare products Regulatory Agency, UK

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4-methyl-4-phenylpentan-2-one

EC Number:

231-007-9

EC Name:

4-methyl-4-phenylpentan-2-one

Cas Number:

7403-42-1

Molecular formula:

C12H16O

IUPAC Name:

4-methyl-4-phenylpentan-2-one

In chemico test system

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/L
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/L
Purity: 94%
Expiry date: 5 years

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
The solubility of the test item in acetonitrile was assessed at a concentration of 100 mM.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019
The positive control was prepared at a concentration of 100 mM.

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of cysteine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

0.703

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

-0.411

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % overall mean depletion

Remarks:

(negative numbers count as zero)

Value:

0.352

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Any other information on results incl. tables

Table 5. Mean peptide depletion of cysteine-containing peptide.

	Peak area (µV.sec)	Peptide concentration^1 (µg/mL)	Peptide depletion^2(%)	Mean depletion ± SD (%)
Positive control	245051	104.38	72.2	72.3 ± 0.10
	243414	103.68	72.4
	244930	104.33	72.2
Test substance	880480	378.50	0.152	0.703 ± 0.20
	870766	374.31	1.25
	967225^3	-	-

 SD: Standard Deviation

¹ Samples prepared at a concentration of 376 µg/mL (0.5 mM)

² Calculated against a mean Reference Control area of 881820 µV.sec(n=6)

³ Result not reported due to loss of solvent during incubation caused by a loose vial cap.

 

Table 6. Mean peptide depletion of lysine-containing peptide.

	Peak area (µV.sec)	Peptide concentration^1 (µg/mL)	Peptide depletion^2(%)	Mean depletion ± SD (%)
Positive control	311319	161.29	58.9	58.4± 0.41
	315241	163.33	58.3
	317420	164.47	58.1
Test substance	759314	394.88	-0.343	-0.411 ± 0.16
	758928	394.67	-0.292
	761248	395.88	-0.598

SD: Standard Deviation

¹ Samples prepared at a concentration of 388 µg/mL (0.5 mM)

² Calculated against a mean Reference Control area of 756720 µV.sec (n=6)

Applicant's summary and conclusion

Interpretation of results:

other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.

Executive summary:

Protein reactivity of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2017).The overall mean percent cysteine and lysine depletion is 0.352 and therefore the test substance is allocated to the “no or minimal reactivity” class (mean % depletion > 0 ≤ 6.38). Thus, the DPRA prediction for protein reactivity is considered negative.