Exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 April 2017 - 13 April 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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]heptan-2-ol

EC Number:

204-712-4

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

124-76-5

Molecular formula:

C10H18O

IUPAC Name:

1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Test material form:

solid

In chemico test system

Details on the study design:

Details on study design:

ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was acetonitrile.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, LLC; Ref. Ac RFAACAA-COOH; batch no 160801; purity 94.82%)
Lysine peptide (supplier RS Synthesis, LLC; Ref. Ac-RFAAKAA-COOH; batch no 160801; purity 94.19%)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5 ± 0.05) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2 ± 0.05) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy.
Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time.
Reference control C: prepared with acetonitrile, the test item and the positive control solvent, in order to check its influence on the peptide stability.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 47.2 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with acetonitrile.
Positive control solution: 38.2 µl of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution with acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 11 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 22 ml of phosphate buffer (100 mM; pH 7.5 ± 0.05).
Lysine solution: 14.8 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 28,6 ml of ammonium acetate buffer (100 mM; pH 10.2 ± 0.05).

TEST SOLUTIONS
Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs).
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
All the replicates were prepared with the same peptide stock solutions.

1 ml of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM Sample): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of sample or solvent for Reference controls + 200 µl of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM Sample): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

The vials were capped and mixed carefully avoiding bubbling, then placed in the HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.
Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples were checked. No precipitate was observed.

Replicates: Each sample was tested 3 times from 3 independent solutions

HPLC ANALYSIS
-Apparatus: Waters e2695 HPLC; Waters 2489 UV detector; Cortecs column C18 2.7 µm ; dimensions 2.1 x 100 mm
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Equilibration of the column: at 30°C with 50% phase A (0.1% (v/v) trifluoroacetic acid in water) and 50% phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for at least 20 minutes before running.
-Volume injected: 7 µl of each sample
-Flow rate: 0.21 ml/min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Duration of re-equilibration to the initial conditions between injections: at least 4 min.
-Analysis sequence:
The analysis was programmed according to the following principles:
The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
The reference controls C were placed at the beginning of each repetition.
The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.
Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.

DEVIATIONS FROM OECD GUIDELINE: No.
The column used has 2.7 µm particle size when the column described in the OECD 442C has 3.5 µm particle size. According to the guideline, the set-up parameters were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides. Proficiency substances recommended in the OECD guideline were performed in these conditions.

Results and discussion

Positive control results:

The depletion mean rate was 73.86% for cysteine peptide and 55.56% for lysine peptide.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of 3 repetitions

Parameter:

other: %depletion in lysine peptide

Value:

2.15

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Mean of 3 repetitions

Parameter:

other: %depletion in cysteine peptide

Value:

1.62

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: Mean of lysine and cysteine peptides

Parameter:

other: mean %depletion in peptides

Value:

1.89

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.511 mM for lysine and 0.502 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 0.86% which is lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.516 mM for lysine and 0.496 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls C was 1.16% which is lower than 15%.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 0.243% for cysteine and 0.43% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 73.86% for cysteine peptide and 55.56% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 1.58% for cysteine and 0.82% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Any other information on results incl. tables

Table 3: Positive control

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	56.06	73.71
Repetition 2	55.31	73.73
Repetition 3	55.32	74.14
SD (Standard Deviation)	0.430	0.243
Mean	55.56	73.86
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100
CV	0.77%	0.33%

Table 4: Test item

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	2.33	0
Repetition 2	1.26	1.71	Mean Depletion %
Repetition 3	2.86	3.16
Mean	2.15	1.62	1.89
SD (Standard Deviation)	0.82	1.58
SD Validity criteria	< 11.6%	< 14.9%

No co-elution occurred of the test item neither with lysine nor with cysteine peptides

Applicant's summary and conclusion

Interpretation of results:

other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.

Conclusions:

The test item shows mean depletion of 2.15% for lysine and 1.62% for cysteine, i.e. an overall average of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for isoborneol as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C ± 2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested 3 times from 3 independent solutions. All validity criteria were fulfilled. The test item shows mean depletion of 2.15% for lysine and 1.62% for cysteine, i.e. an overall average of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.