Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl propionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr 2016 to 4 Aug 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Remarks:

Not specified in report, however, according to the website of the testing laboratory, all assays are performed under GLP

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

EC Number:

204-727-6

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

Cas Number:

125-12-2

Molecular formula:

C12H20O2

In chemico test system

Details on the study design:

SOLUBILITY TEST
A solubility test was performed for each test article in order to determine an appropriate solvent that completely dissolved the test article at a 100 mM concentration.

PEPTIDE PREPARATION
- Custom synthetic peptides of cysteine or lysine (containing phenylalanine to aid in detection) were used. The purity of each peptide was 90-95%. Peptide samples were newly prepared for each sample set, and a single preparation of the peptide was used throughout the sample set. - The cysteine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.5 phosphate buffer.
- The lysine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 10.2 ammonium acetate buffer.
- The peptide solutions were gently mixed on the shaker.

PEPTIDE STANDARDS
- A set of serially diluted standards were prepared for each peptide. These standards were prepared by diluting the peptide solutions in dilution buffer (20% acetonitrile in either phosphate or ammonium acetate buffer).
- Six standards were prepared at concentrations of 0.534- 0.0167 mM. A seventh standard was prepared containing only dilution buffer.
- Approximately 1 mL of each standard was pipetted into the appropriate prelabelled autosampler vials.

CONTROLS
- The positive control used in this assay was cinnamic aldehyde prepared at a concentration of 100 mM. The positive control was reacted with the peptides in the same fashion as the test article.
- Reference controls were prepared for each solvent used in the assay. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). These controls consist of the solvent (acetonitrile) reacted with the peptide in the absence of test article.
- Coelution controls were prepared for each test article. The coelution controls consisted of the test articles without the peptide. The purpose of the coelution controls were to determine if the test article elution from the HPLC column overlapped with the peptide elution.

REACTION MIXTURE PREPARATION
- Immediately prior to testing, each test article was diluted in the appropriate solvent to yield a 100 mM test article concentration. The test articles were mixed as determined during the solubility test (vortexing). The final dosing solutions were prepared for each test article, positive control, and reference control in the prelabelled autosampler vials.
- Triplicate samples were prepared for each test article and control. Single samples were prepared for the coelution controls.
- The make-up of the reaction mixtures was as follows: 1:10 Ratio Cysteine peptide (0.5 mM Peptide, 5 mM test substance) and 1:50 ration Lysine Peptide (0.5 mM Peptide, 25 mM test substance).

HPLC SETUP AND OPERATION
- The separations module used in this assay was a Waters 2690/5 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test articles and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The dimensions of the column used were 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least 2 hours before the start of the assay. To prime the column, equal parts of mobile phase A (0.1 % TFA in HPLC grade water) and mobile phase B (0.08% TFA in HPLC grade acetonitrile) were passed through the column.
- Once the column was equilibrated and the samples were prepared, the autosampler vials were placed intro the designated location of the separation module carousels. The samples were incubated in the dark at room temperature for 24 ± 2 hours.
- A gradient elution was used in this assay. The mobile phase changed from 10 to 25% acetonitrile over a 10 minute period to allow for optimal separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections.
- The Empower PDA software was used to convert the absorbance data from the UV detector into chromatograms of intensity versus retention time for each sample and control. At the end of the run, each chromatogram was integrated in order for the software to calculate the area under the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible to determine which peaks in the chromatograms represented the peptides and use the areas under those peaks for the subsequent calculations.

CALCULATIONS
- The peak area calculations were obtained from the Empower PDA software and entered into a spreadsheet. Standard curves of peak area versus concentration were prepared for each set of peptide standards. The concentrations were calculated for each sample using peak area and the equation for the appropriate standard curve. The peak area for each sample was substituted for y and the equation was solved for x to determine the peptide concentration of the sample.
- The percent depletion was calculated for each test article sample and the positive control Samples: % peptide depletion = (1 – [test article or positive control peptide peak area / mean peptide peak area of reference control C] x 100). Reference control C consisted of three replicates of acetonitrile and peptide run concurrently with the test article samples.

Results and discussion

Positive control results:

The positive control substance Cinnamic Aldehyde caused a mean peptide depletion of 75.4 and 59.8% for Cysteine and Lysine, respectively.

In vitro / in chemico

Results

Results 1

Parameter:

other: Mean Peptide depletion of Cysteine and Lysine (%)

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Mean Percent Peptide Depletion (%)

Parameter:

other: Cysteine reactivity

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: Mean Percent Peptide Depletion (%)

Parameter:

other: Lysine reactivity

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

- The substance was found to be soluble in acetonitrile with vortexing for approxirnately 1 minute.

Applicant's summary and conclusion

Interpretation of results:

other: Negative

Conclusions:

Isobornyl acetate is negative in the DPRA (OECD TG 442C).

Executive summary:

The Direct Peptide Reactivity Assay (similar to OECD TG 442C) was used in a weight-of-evidence approach to assess the skin sensitizing potential of the test substance. Synthetic peptides containing cysteine or lysine were reacted with 100 mM test article for 24± 2 hours in a ratio of 1:10 (cysteine) or 1:50 (Lysine). Acetonitrile was used as vehicle and Cinnamic aldehyde was used as positive control. After the incubation period the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection. The positive control resulted in 75.4 and 59.8% depletion of the respective peptides. The test substance gave a mean peptide depletion of 0.4% and 0.0% for Cysteine and Lysine, respectively. The mean peptide depletion of Cysteine and Lysine was 0.2%. Based on these results, the test is considered negative.