Reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 -18 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

yes

Remarks:

No information on stability, precision and co-elution controls provided. No information on stability/solubility of test substance after preparation of peptide depletion samples and after 22 h incubation.

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

EC Number:

945-924-3

Molecular formula:

C13H18O2

IUPAC Name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

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: 90 - 95%
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:

0.906 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

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:

-1.68 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % overall mean depletion

Value:

0.453 %

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

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 (µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	902257	381	0.608	0.906 ± 0.28
	899025	379	0.964
	897389	378	01.15
Positive control	244716	103	73.0	73.0 ± 0.35
	245752	104	72.9
	244042	103	73.1

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

**: Calculated against a mean reference control peak area of 940000 μV.sec (n=6)

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

	Peak area (µV.sec)	Peptide concentration (µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	792447	410	-3.34	-1.68 ± 1.81
	782216	405	-2.00
	764584	396	0.295
Positive control	337396	175	56.0	56.3 ± 1.54
	338682	176	55.8
	329160	171	57.1

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

**: Calculated against a mean control peak area of 766850 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.