2,3-dihydro-2,2,6-trimethylbenzaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From February 06, 2019 to March 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

EC Number:

204-133-7

EC Name:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

Cas Number:

116-26-7

Molecular formula:

C10H14O

IUPAC Name:

2,6,6-trimethylcyclohexa-1,3-diene-1-carbaldehyde

Test material form:

liquid

In chemico test system

Details on the study design:

Preparation if test solution: Accurately weighed 150.20 mg of Safranal in 10.0 mL volumetric flask, and dissolved in Acetonitrile : Milli Q water (1:1 v/v) and volume was made up to 10 mL with Acetonitrile: Milli Q water ( 1:1 v/v).
Accurately weighed 150.19 mg of Safranal in 10.0 mL volumetric flask, and dissolved in Acetonitrilc : Milli Q water (1:1 v/v) and volume was made up to 10 mL with Acelonitrile : Milli Q water (1:1 v/v).
Preparation of standard solutions: - Cysteine peptide: 0.669 mM stock solutions of cysteine peptide were prepared by diluting 2.51 mg of cysteine peptide with 5.0 mL of Sodium phosphate buffer of pH 7.5.
- Lysine peptide: 0.668 mM stock solutions of lysine peptide was prepared by diluting 2.59 mg of lysine peptide with 5.0 mL of 100 mM ammonium acetate buffer pH 10.2.

Peptide and test item (1:10 Dilution)
- Cysteine Peptide: a volume of 375µL of cysteine stock solution, 400 µL of phosphate bufler pH 7.5, 25 µLof sample stock solution and 200 µL of acetonitrile were added to HPLC vial.
- Lysine Peptide: a volume of 375µL of lysine stock solution, 400 µL of ammonium acetate bufler pH 10.2, 25 µLof sample stock solution and 200 µL of acetonitrile were added to HPLC vial.
Peptide and test item (1:50 Dilution)
- Cysteine Peptide: a volume of 375µL of cysteine stock solution, 300 µL of phosphate bufler pH 7.5, 125 µLof sample stock solution and 200 µL of acetonitrile were added to HPLC vial.
- Lysine Peptide: a volume of 375µL of lysine stock solution, 300 µL of ammonium acetate bufler pH 10.2, 125 µLof sample stock solution and 200 µL of acetonitrile were added to HPLC vial.
HPLC vials were gently vortexed and incubated for 24 hours at 25°C ± 2.5°C in the (dark) prior to HPLC analysis. Preparation was done in triplicates and single HPLC run was performed for each preparation.

Preparation of Control: A volume of 375 µL of cysteine stock solution, 425 µL phosphate buffer pH 7.5 and 200 µL of acetonitrile were added to HPLC vial.
A volume of 375 µL of lysine stock solution, 425 µL ammonium acetate buffer pH 10.2 and 200 µL of acetonitrile were added lo HPLC vial.
HPLC vials will be gently vortexed and incubated for 24 hours at 25°C ± 2.5°C in the dark place prior to HPLC analysis. Preparation was done in triplicates and single HPLC run will be performed for each preparation.

Prepation of Linearity Standards for Cysteine Peptide:
• A volume of 25 µL of cysteine stock solution, 925 µL phosphate buffer pH 7.5 and 50 µL of acetonitrile were added to HPLC vial (0.0167 mM).
• A volume of 50 µL of cysteine stock solution, 900 µL phosphate buffer pH 7.5 and 50 µL of acetonitrile were added to HPLC vial (0.0334 mM).
• A volume of 100 µL of cysteine stock solution, 850 µL phosphate buffer pH 7.5 and 50 µL of acetonitrile were added to HPLC via l (0.0669 mM).
• A volume of 200 µL of cysteine stock solution, 750 µL phosphate buffer pH 7.5 and 50 µL of acetonitrile were added to HPLC vial (0. 1337 mM).
• A volume of 200 µL of cysteine stock solution, 275 µL phosphate buffer pH 7.5 and 25 µL of acetonitrile were added to HPLC vial (0.2674mM).
• A volume of 400 µL of cysleine stock solution, 75 µL phosphate buffer pH 7.5 and 25 µL of acetonitrile were added to HPLC vial (0.5348 mM).
Each standard was injected and a linearity curve was prepared using regression analysis.The correlation coefficient, slope and intercept were calculated.

Prepara tion or Linearity Standard for Lysine Peptide
• A volume of 25 µL of lysine stock solution, 925 µL ammonium acetate buff'er pH 10.2 and 50 µL of acetonitrile were added to HPLC vial (0.0 167 mM).
• A volume of 50 µL of lysine slock solution, 900 µL ammonium acetate buffer pH 10.2 and 50 µL of acetonitrile were added to HPLC vial (0.0334mM).
• A volume of 100 µL of lysine stock solution, 850 µL ammonium acetate buffer pH 10. 2 and 50 µL of acetonitrile were added to HPLC vial (0.0668 mM).
• A volume of 200 µL of lysine stock solution, 750 µL ammonium acetate buffer pH 10.2 and 50µL of acetonitrie were added to HPLC vial (0.1355 mM).
• A volume of 200 µL of lysine stock solution, 275 µL ammonium acetate buffer pH 10.2 and 25 µL of acetonitrile were added to HPLC vial (0.2670 mM).
• A volume of 400 µL of lysine stock solution, 50 µL ammonium acetate buffer pH 10.2 and 25 µL of acetonitrile were added to HPLC vial (0.5341 mM ).
Each standar was injected and a linearity curve was prepared using regression analysis. The correlation coefficient, slope and intercept were calculated.

Analysis: All prepared samples were loaded to HPLC sequence and chromatograms were determined at 220 run. Peptide reactivity with the test item was reported as percent peptide depletion, which was determined as the reductionof the peptide concentration in the samples relative to the average concentration of the controls.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine (1:10)

Parameter:

other: Mean of Cysteine depletion

Value:

90.63

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Lysine (1:50)

Parameter:

other: Mean of Lysine depletion

Value:

20.81

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The mean value of the percent cysteine and lysine depletion was calculated for the test item. From the mean value of the percent cysteine and lysine depletion, the reactivity class was classified and the skin sensitivity was predicted.

Any other information on results incl. tables

Evaluation Method:

Mean of Cysteine and Lysine depletion	Reactivity Class	Prediction
0% < Mean % Depletion < 6.38%	Minimal Reactivity Non- Sensitiser	Negative
6.38% < Mean % Depletion < 22.62%	Low Reactivity Sensitiser	Positive
22.62% < Mean % Depletion < 42.47%	Moderate Reactivity Sensitiser
42.47%< Mean % Deplet ion < 100 %	High Reactivity Sensitiser

Results:

Particulars	Sample Code	Sample Peak Area (mAU)	Mean Sample Peak Area (mAU)	Percent Peptide Depletion	Mean Percent Peptide Depletion
Cysteine (1:10)	Control R1	1144.19	12 16.98	-	-
	Control R2	1260.77
	Control R3	1245.99
	Sample Rl	119.62	-	90.17	90.63
	Sample R2	108.2 2		91.11
	Sample R3	I14.21		90.62
Lysine (1:50)	Control R1	680.48	704.435	-	-
	Control R2	724.98
	Control R3	707.85
	Sample RI	544.9	-	22.65	20.81
	Sample R2	553.78		21.39
	Sample R3	574.76		18.41
Average Percent Depiction					55.72
Prediction					High Reactivitv

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The percent peptide depletion was 90.63% for cysteine peptide solution and 20.81% for lysine peptide solution. The mean value of the percent cysteine ad lysine depletion was 55.72%. Therefore, the reactivity class of test item was classified to " High Reactivity Sensitizer" and skin sensitivity was predicted as " Positive" in these testing conditions.

Executive summary:

The objective of the study was to screen the test item Safranal for skin sensitizing potential by direct peptide reactivity assay (DPRA).

The test item dissolved in acetonitrile was mixed with cysteine peptide solution (1:10) and lysine peptide solution (1:50) respectively, was incubated at25±2.5°C for 24 hours. After 24hours of incubation the reaction solutions were analysed by high performance liquid chromatography and peak area for each peptide was determined. Percent cysteine and percent lysine peptide depletion, and the average value of the percent peptide depletions were calculated.

Consequently, the percent peptide depletion was 90.63% for cysteine peptide solution (1:10) and 20.81% for lysine peptide solution (1:50). The mean value of the percent cysteine and lysine depletion was 55.72%.Therefore,the reactivity class of test item was classified to" High Reactivity sensitizer"and skin sensitivity was predicted as "Positive".