Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

EC Number:

915-335-6

Molecular formula:

C11H20O2 C10H20O

IUPAC Name:

Reaction Mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

Test material form:

liquid

In chemico test system

Details on the study design:

- Assessment of Test Item Solubility: The solubility of the test substance was assessed in acetonitrile.
- 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).
- Preparation of Peptide Calibration Standards: 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.
- Preparation of Stability Controls and Precision Control: Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile.
- Preparation of Positive Control Solution and Test Item Stock Solution: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of test substance was also prepared in acetonitrile.
- Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Triplicate solutions each of the positive control and test substance stock solutions were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM test substance. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
- Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: Triplicate solutions each of the positive control and test substance stock solution were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM test substance. For the co-elution control, buffer solution was used in place of the Lysine stock solution.
- Incubation: The appearance of the test substance and positive control samples in the HPLC vials was documented following preparation and then the vials were 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.
- Analysis: The concentration of both the Cysteine and Lysine peptides in the presence of test substance and the associated positive controls was quantified by HPLC using UV detection.
- Instrumentation Parameters: High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector; Column: Agilent Zorbax SB C18, 3.5μm, 100 × 2.1 mm; Guard column: Phenomenex AJO4286; Column temperature: 30°C; Sample temperature: 25°C; Mobile Phase (MP) A: 0.1% TFA in Water; Mobile Phase (MP) B: 0.085% TFA in ACN; Gradient (MP A (%):MP B (%)): 0 min: 90:10; 20 min: 75:25; 21 min: 10:90; 23 min: 10:90; 23.5 min: 90:10; 30 min: 90:10; Flow rate: 0.35 mL/minute; Stroke volume: 25 μL; Detector wavelength: UV, 220 nm; Injection volume: 2 μL (slow draw rate); Run time: 30 minutes; Approximate retention time (Cysteine): 11 minutes; Approximate retention time (Lysine): 7 minutes
- Calculations: The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation: % Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100)/Mean Peptide peak area of reference control samples B)

Results and discussion

Positive control results:

69.3% depletion (SD 0.32%, n = 3) and 57.7% depletion (SD 0.21%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Mean Cysteine depletion

Value:

0.74

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Parameter:

other: Mean Lysine depletion

Value:

-0.028

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Solubility Assessment: Solubility of Dimyrcetol was achieved at a nominal concentration of 100 mM in acetonitrile.
- There were no co-elution peaks in either the Cysteine or Lysine assay.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls of both peptides were met (0.510 mM, CV 2.30%, n = 6 and 0.506 mM CV 0.55%, n = 6, for cysteine and lysine, respectively).
- Acceptance criteria met for positive control: yes, see above
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.65% (n=3) and 0.54% (n=3), respectively, for cysteine and lysine depletion by the test item. S

Applicant's summary and conclusion

Interpretation of results:

other: DPRA is negative

Conclusions:

It can be concluded that this DPRA test is valid and the test substance is negative because the substance showed mean depletion of cysteine and lysine of -0.74 and -0.0280%, respectively, which is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. There was no to minimal reactivity of both peptides in the presence of the test substance. With an overall depletion of peptide of 0.370% (0.74% and -0.0280% for cysteine and lysine, respectively), the reactivity of the test substance is henceforth classified as “no or minimal” therefore the DPRA prediction is negative.