Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb to March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

EC Number:

261-118-8

EC Name:

Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

Cas Number:

58096-47-2

Molecular formula:

C16H26O2

IUPAC Name:

decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate

Test material form:

liquid

Specific details on test material used for the study:

Decahydro-1,1,7-trimethyl-3a,7-methano-3aH-cyclopentacyclooct-3-yl formate (Product Name: Clovanyl-3-Formate/Caryolan)

In chemico test system

Details on the study design:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25±2.5ºC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by High Performance Liquid Chromatography (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 (Section 11) which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Results and discussion

Positive control results:

The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.89% which shows that the positive control is a sensitizer with high reactivity, according to cysteine 1:10/lysine 1:50 prediction model, in Direct Peptide Reactivity Assay (DPRA) confirming the sensitivity of the test.

In vitro / in chemico

Results

Key result

Parameter:

other: Mean % peptide depletion

Value:

1

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:

All criteria for test chemical were met for the run to be considered valid.
a) The maximum standard deviation (SD) of sample replicates was 9.76% (limit < 14.9%) for the percent cysteine depletion and 1.27% (limit <11.6%) for the percent lysine depletion
b) For each solvent used, the mean of the peptide concentrations of the three appropriate Reference Controls C was 0.47 mM and 0.51 mM for cysteine and lysine peptide respectively (range 0.50±0.05 mM).

Any other information on results incl. tables

Table: Reactivity Class Classification of Test Item and Positive Control as per Cysteine 1:10/Lysine 1:50 Prediction Model

Sample ID/ Code	Name of the chemical	Mean % of Cysteine Peptide Depletion	Mean % of Lysine Peptide Depletion	Mean % peptide depletion	Reactivity class	DPRA prediction
CA	Cinnamic aldehyde	71.45	56.33	63.89	High Reactivity	Positive
A076-01	Clovanyl-3-Formate (CARYOLAN)	0	2.00	1.00	No or Minimal Reactivity	Negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean of cysteine and lysine peptide depletion by the test item [Clovanyl-3-Formate (CARYOLAN)] was 1.00% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, test item [Clovanyl-3-Formate (CARYOLAN)] was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).

Executive summary:

Skin sensitisation test byin chemicotest method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate the skin sensitisation potential of the test item, Clovanyl-3-Formate (CARYOLAN) and to classify as skin sensitiser or non-skin sensitiser.

Solubility check was performed for the test item before test sample analysis. The test item, Clovanyl-3-Formate (CARYOLAN), was found to be soluble in acetonitrile and formed a homogenous solution at 100 mM concentration.

On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.  

Test item and positive control solutions prepared at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC as per section 9 to measure the peptide depletion.

Cysteine and lysine peptide Percent Depletion Values are then calculated from peptide peak areas obtained from the HPLC analysis. The data of test sample analysis meeting all the acceptance criterion as per OECD 442C were only considered for reporting. The test item Clovanyl-3-Formate (CARYOLAN) showed 0% mean cysteine peptide depletion and 2.0% mean lysine peptide depletion. Under the same circumstances positive control (cinnamaldehyde) showed 71.45% and 56.33% mean cysteine and lysine peptide depletion.

To classify test item as sensitizers and non- sensitizers, the cysteine 1:10/lysine 1:50 prediction was used. The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.89% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [Clovanyl-3-Formate (CARYOLAN)] was 1.00% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, test item [Clovanyl-3-Formate (CARYOLAN)] was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).