2-methyl-4-phenylbutan-2-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 April 2017 - 13 June 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))

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-methyl-4-phenylbutan-2-ol

EC Number:

203-074-4

EC Name:

2-methyl-4-phenylbutan-2-ol

Cas Number:

103-05-9

Molecular formula:

C11H16O

IUPAC Name:

2-methyl-4-phenylbutan-2-ol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company LTD; CP008-170101
- Expiration date of the lot/batch: 30 June 2018
- Purity: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature, protected from light and humidity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was diluted in the selected vehicle (acetonitrile) at 100 mM. This formulation had the aspect of a colorless liquid. The formulation was used just after its preparation.

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 442E were also performed. The assay was run once, in triplicate.

Assay Controls:
- The vehicle was acetonitrile. DMPEC was diluted at 100 mM in acetonitrile.

- The positive control was cinnamaldehyde (CAS No. 104-55-2) at 100mM

- Co-elution control samples
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

- Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent specified in Section 2.4.1.2 Reference control samples preparation. These samples were used to:
-reference control A: check the accuracy of the calibration curve for peptide quantification,
-reference control B: check the stability of the peptide during analysis,
-eference control C: check that the solvent did not impact the percentage of peptide depletion


2.4 DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.

2.4.1 Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

2.4.1.1 Co-elution control samples preparation
For the co-elution control with cysteine peptide:
50 µL of test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile.

For the co-elution control with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

2.4.1.2 Reference control samples preparation
2.4.1.2.1 Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

2.4.1.2.2 Reference control C samples
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference control C prepared with cysteine peptide:
50 µL of vehicle (acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reference control C prepared with lysine peptide:
In parallel, 250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

2.4.1.3 Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.4.1.4 Test item samples preparation
For the reactivity of test item with cysteine peptide:
50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.

For the reactivity of test item with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.4.2 Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system.

At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation (see § 3 Results).

Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

2.4.3 Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

2.4.4 HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
one blank sample (peptide dilution buffer),
-one calibration curve injected at the beginning of the analytical batch,
-three reference control A samples,
-the co-elution control sample,
-three reference control B samples,
-reference control C samples (replicate 1),
-positive control sample (replicate 1),
-test item study sample (replicate 1).
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
-three reference control B samples.

Results and discussion

Positive control results:

For the cysteine peptide, the mean depletion value was 96.24%, For the lysine peptide, the mean depletion value was 63.28 %.
The mean of the percent cysteine and percent lysine depletions was equal to 79.76%. This gives a classification of High reactivity.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: DMPEC

Parameter:

other: Cysteine - mean depletion (%)

Value:

0.75

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Run / experiment:

other: DMPEC

Parameter:

other: Lysine - mean depletion (%)

Value:

0.01

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Run / experiment:

other: DMPEC

Parameter:

other: Mean (Cysteine/Lysine depletions, %)

Value:

0.38

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in the test item samples incubated with the cysteine and lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive samples and reference control samples prepared in acetonitrile were also centrifuged in the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected into the HPLC/UV system.
For the other samples (co-elution samples prepared with the lysine and cysteine dilution buffers), the vials were directly transferred into the HPLC/UV system


ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 442E were also performed. Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item Dimethyl phenyl ethyl carbinol was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the peptides at the end of the incubation.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; 45133TIR), DMPEC (99.7%) in acetonitrile was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control sample C in acetonitrile. The positive control was cinnamaldehyde.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values calculated for each peptide were 0.75% for the cysteine peptide and 0.01% for the lysine peptide. The mean of the percent cysteine and percent lysine depletions was equal to 0.38%. However, since precipitate was observed at the end of the incubation with the peptides, the peptides depletion may be underestimated.  Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no or minimal reactivity. Therefore, the DPRA prediction for DMPEC is considered as negative, though with limitations as the test item precipitates with the peptides at the end of the incubation.

The results from this assay will be combined with the results from the OECD 442D and OECD 442E assays for a final prediction on the skin sensitisation properties of DMPEC.