2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-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:

06 October 2020 - 12 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442C and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on 04 February 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Certificate for GLP compliance dated on 1st of October 2019.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

EC Number:

815-500-1

Cas Number:

1853175-99-1

Molecular formula:

C15 H26 O2

IUPAC Name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

Test material form:

liquid

Details on test material:

- Physical state: Colorless liquid
- Storage condition of test material: Store in cool (ambient temperature) and dry place

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS

- Preparation of the peptide stock solutions: 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 serial dilution of 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 the test chemical solutions: the test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation was colorless limpid solution and was used just after its 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).

- Preparation of the positive controls, reference controls and co-elution controls:
* Precision controls (Reference Control A, n=3) and stability controls (Reference Control B, n=6), of both peptides were prepared at a peptide concentration of 0.5 mM. Reference Control A was used to assess the precision of injection, while Reference Control B was used to verify the stability of the peptide response over time and as a comparison point to determine depletion of the peptide in test samples.
* The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
* Accurate volume aliquots of the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of the positive control. For the co-elution controls, buffer solution was used in place of the Cysteine stock solution.
* Accurate volume aliquots of the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of the positive control. For the co-elution controls, buffer solution was used in place of the Lysine stock solution.

INCUBATION:

Incubation conditions: the appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run.
Before initiation of the run the appearance of the samples in the vials was assessed visually to check the absence of precipitate and documented again.

PREPARATION OF THE HPLC

- Instrumentation Parameters:
The HPLC/UV method used for the samples analysis according to the recommendations of the OECD guideline No. 442C is described in Covance internal analytical method and is summarized in the table below:
* HPLC: Waters Alliance 2695 separation module and 2487 dual wavelength detector
* Analytical Column: Agilent Zorbax SB C18, 100 x 2.1 mm, 3.5 μm
* Guard column: Phenomenex AJO4286
* Column temperature: 30°C
* Sample temperature: 25°C
* Mobil phase: Mobile phase A: 0.1% TFA in water / Mobile phase B: 0.085% TFA in Acetonitrile.
* Gradient:
Time % Mobile phase A % Mobile phase B
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
* Flow rate: 350 µL/minute
* Strocke volume: 25 µL
* Detector wavelength: UV, 220 nm
* Injection volume: 2 µL
* Retention times: Cysteine-peptide: approx. 11 minutes / Lysine-peptide: approx. 6.8 minutes
* Total analysis time: 30 minutes

- Verification of the suitability of the HPLC for test chemical and control substances: yes.
* Monitor retention time and tailing factor for each analytical run.
* Retention time and tailing factor should not deviate beyond the limits set below for the highest calibration standard (standard 6 of each peptide).
* Limits for system suitability: lysine peptide:
Retention time (min) +/- 10%: 6.84 (Nominal), 6.16 (Minimum), 7.52 (Maximum)
Tailing factor +/- 10%: 1.50 (Nominal), 1.35 (Minimum), 1.65 (Maximum)

* Limits for system suitability: Cysteine peptide:
Retention time (min) +/- 10%: 11.26 (Nominal), 10.13 (Minimum), 12.39 (Maximum)
Tailing factor +/- 10%: 1.43 (Nominal), 1.29 (Minimum), 1.57 (Maximum)

DATA EVALUATION:

- Cys and Lys peptide detection wavelength: 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 or C].

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 70.6% and SD = 0.17%
- Result of positive control samples in lysine assay: Mean % depletion = 57.8% and SD = 0.61%
Mean depletion rate of Cinnamaldehyde: 64.2%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

93.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: High reactivity

Remarks:

DPRA Predicition: Positive.

Results 2

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: No or minimal reactivity

Remarks:

DPRA Prediction: Negative.

Results 3

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

46.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: High reactivity

Remarks:

DPRA Prediction: positive

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media.
In Lysine assay samples and the co-elution control, there was evidence of phase separation, with a distinct “lens” of separated material noted on the solution surface. The dissolution control (test item in acetonitrile) was a clear colourless solution.
In Cysteine assay samples, a cloudy appearance was noted, attributed to the presence of a fine white precipitate. In contrast, the dissolution control was a clear colourless solution, while the co-elution control showed evidence of phase separation similar to the Lysine assay samples.
The precipitate present in Cysteine assay samples may be attributed to reaction products of the peptide and the test substance.

There were no co-elution peaks in either the Cysteine or Lysine assays.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: not specified.

Any other information on results incl. tables

Solubility results

The solubility of the test item in acetonitrile at a nominal concentration of 100 mM was achieved.
Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media. 

Reactivity Assessment: 
The DPRA prediction and the reactivity of the test substance based on the overall mean and the individual depletion values in the Cysteine peptide and the Lysine peptide is presented in Table 7.4.1/1 and Table 7.4.1/2. 

 

Applicant's summary and conclusion

Interpretation of results:

other: Positive in the DPRA assay.

Conclusions:

Under the experimental conditions of this study, the mean depletion of both peptides was 46.9%. Therefore, the test item was considered to have high peptide reactivity and hence it is predicted by DPRA to be positive in terms of being a potential skin sensitizer.

Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay using the validated DPRA method (Covance analytical method FIA/M101/15, based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) and in compliance with GLP.

 

 The reactivity of the test item was evaluated by monitoring peptide depletion following an approximate 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for a minimum of 22 hours in glass autosampler vials, protected from light and set at 25°C. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm.

The test item was dissolved at 100 mM in acetonitrile without sonication.

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.

 

The Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media.

In Lysine assay samples and the co-elution control, there was evidence of phase separation, with a distinct “lens” of separated material noted on the solution surface. The dissolution control (test item in acetonitrile) was a clear colourless solution.

In Cysteine assay samples, a cloudy appearance was noted, attributed to the presence of a fine white precipitate. In contrast, the dissolution control was a clear colourless solution, while the co-elution control showed evidence of phase separation similar to the Lysine assay samples. The precipitate present in Cysteine assay samples may be attributed to reaction products of the peptide and the test substance.

As described in the OECD Guideline 442C, if a precipitate is observed immediately upon addition of the test chemical solution, due to low aqueous solubility of the test chemical, although one cannot be sure how much test chemical remained in the solution to react with the peptide, in such case, a positive result can still be used. Therefore, the cysteine depletion values is considered as valid.

The cysteine depletion value was 93.4%, the lysine depletion value was 0.3% and the mean of the cysteine and lysine depletion values was 46.9%. All acceptance criteria were met.

 

Under the experimental conditions of this study, the test item was considered to have high peptide reactivity and hence it is predicted by DPRA to be positive in terms of being a potential skin sensitizer.