[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Mar - 12 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and OECD Guideline 442C. However, due to the limited solubility of the test substance precipitation was observed after 24 h incubation. Since a negative result was obtained for the test substance in this study, no conclusion on the lack of reactivity can be drawn according to OECD Guideline 442C.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

EC Number:

216-365-6

EC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Cas Number:

1565-76-0

Molecular formula:

C11H22O

IUPAC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

In chemico test system

Details on the study design:

TEST SYSTEM
- Source: GenScript, Piscataway, US and RS Synthesis, Louisville, US
- Specification of the peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

TEST METHOD
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 h incubation with the test substance at 25 ± 2.5°C. The synthetic peptides contain phenylalanine to aid in the detection. 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 a 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 SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile. The solvent was chosen because the test substance was soluble in the vehicle.

INCUBATION CONDITIONS
- Temperature: 25 ± 2.5 °C
- Duration: 24 ± 2 h

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent HP 1100 with DAD (Software: Dionex Chromeleon)
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column „Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 25
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL

NUMBER OF REPLICATIONS: samples were prepared in triplicates for each peptide

EXPERIMENTAL PROCEDURE
- Test substance solubility: Prior to the assay the solubility of the test substance was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.
- Preparation of calibration samples: The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution (please refer to Table 1). The analysis of the calibration samples was started before analysis of the test-substance samples.
- Preparation of test substance samples: The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 h. The HLPC analysis of the batch of samples started about 24 h after sample preparation and the analysis time itself did not exceed 30 h.
- Preparation of vehicle controls: Several acetonitrile controls were prepared in triplicates in the same way as the test substance samples described above but with acetonitrile instead of the test substance. Set A was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in acetonitrile.
- Preparation of the co-elution control: The co-elution control was prepared in the same way as the test substance samples described above but without the peptide. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples.

Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. As the samples were visually turbid or displayed precipitates they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles.

Positive control: ethylene glycol dimethacrylate

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean C-peptide depletion

Value:

-0.7 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Parameter:

other: mean K-peptide depletion

Value:

-0.97 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
No co-elution of test substance and peptides was noticed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: The acceptance criteria were met with the exception of the vehicle control A of the K-peptide samples which is not availbale due to a technical error. However, the test is considered to be valid despite the lack of the performance control, as all other control samples (samples B and C) met the acceptance criteria.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were homogeneous emulsions immediately after preparation. After 24 h precipitates were noticed in the samples of the C-peptide. The samples of the K-peptide were visually homogeneous emulsions after 24 h. Thus all samples were centrifuged prior to HPLC analysis.

COMPARISON WITH HISTORICAL CONTROL DATA
Historic control values of negative and positive controls, gathered over an appropriate time period, demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Any other information on results incl. tables

Table 6. Mean peptide depletions of cysteine, lysine and both peptides.

	Cysteine-Peptide		Lysine-Peptide		Mean of both depletions (%)
	Mean depletion (%)	SD	Mean depletion (%)	SD
Test substance	-0.70	1.60	-0.97	0.22	0.00
Positive control	55.05	5.56	14.50	1.48	34.77

 

The mean C-peptide depletion, caused by the test substance was determined to be -0.70%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.97%.

 

Negative depletions were considered to be 0 for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the observed results it was concluded that the test substance shows a minimal chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. According to OECD Guideline 442C a negative result should be considered inconclusive in this case.

Executive summary:

It was concluded that Menthylmethylether shows a minimal chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive.