1,3,3-trimethylnorbornan-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 February 2018- 02 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 (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,3,3-trimethylnorbornan-2-one

EC Number:

232-107-5

EC Name:

1,3,3-trimethylnorbornan-2-one

Cas Number:

7787-20-4

Molecular formula:

C10H16O

IUPAC Name:

1,3,3-trimethylnorbornan-2-one

Test material form:

liquid

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, Louisville KY, USA; Ref. Ac RFAACAA-COOH; batch no 170809, 171229)
Lysine peptide (supplier RS Synthesis, Louisville KY, USA; Ref. Ac-RFAAKAA-COOH; batch no 170809, 161121)

CONTROLS
Positive control: Cinnamic Aldehyde (CAS 104-55-2; batch no MKBX8146V, Sigma Aldrich, purity 98.9%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2) for lysine peptide.
Reference control A, B and C were prepared by adding 250 µL of acetonitrile to 750 µL of 0.667 mM Lysine peptide prepared in 100 mM Ammonium acetate buffer or 0.667 mM cysteine peptide prepared in 100 mM phosphate buffer.
Reference control A was used to verify the accuracy of the calibration curve for peptide quantification. Reference control A was injected in triplicates.
Reference control B was injected in triplicates each both in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Reference control C was included in every assay run together with the samples in triplicates. They were used to verify that the solvent does not have any impact on the percent peptide depletion. The Reference Controls C was used to calculate Percent Peptide Depletion by the test item.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item and positive control were dispensed using micropipette into clean and dry glass vials. 100 mM stock solution of test item and positive control (Cinnamic aldehyde) was prepared fresh, immediately before use.
Test item solution: Test item stock solution of 100 mM was prepared by dispensing 35.3 µL of test item into a vial and dissolved by adding 1964.7 µL of acetonitrile and mixed well.
Positive control solution: Positive control of 100 mM stock solution was prepared by dispensing 26.5 µL into a vial followed by adding 1973.5 µL of acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: Cysteine peptide Ac-RFAACAA-COOH, 0.667mM, 0.501 mg/mL: Using an electronic balance, Cysteine peptide was weighed and dissolved in appropriate volume of phosphate buffer.
Lysine solution: Lysine peptide Ac-RFAAKAA-COOH, 0.667mM, 0.518 mg/mL: Using an electronic balance, Lysine peptide was weighed and dissolved in appropriate volume of ammonium acetate buffer.

TEST SOLUTIONS
For each run, test item stock and positive control solutions were prepared before the start of sample preparation for analysis.
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
Test samples were prepared in triplicates for both peptides.
Both cysteine and lysine peptides were prepared on the same day once and remaining experiments peptides were tested individually.
All the replicates analysed in the same run 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 test chemical): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of test chemical solution or solvent for Reference controls + 200 µl of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM test chemical): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of test chemical solution or solvent for Reference controls.

Vials were capped, vortexed to mix and placed in (dark) at 25ºC for 24 hours. HPLC analysis of the batch of samples was started after 24 hours the test item addition to the peptide solution.

HPLC ANALYSIS
Apparatus:
Column: Agilent Zorbax SB-C18 2.1 mm X 100 mm X 3.5 micron
Detector: Photodiode Array detector or Fixed Wavelength Absorbance detector with 220 nm signal for quantification.
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: not specified.
Volume injected: 10 µl of each sample
Flow rate: 0.35 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: Not specified.
Analysis sequence:
Two separate analysis sequences were prepared as below:
Analysis sequence 1: Calibration standards (Standard curve), Reference Controls A and Co-elution Controls.
Analysis sequence 2: Reference Controls B and sets of replicates of Reference Controls C, Positive Control and test item.
The first analysis sequence was timed to complete prior to the end of the 24 hour incubation and the second analysis sequence was timed to assure that the first sample starts 24 ± 2 hours after the test item was mixed with the peptide solution.
The difference between the first replicate analysis and the last replicate analysis was not more than 30 hours.

DEVIATIONS FROM OECD GUIDELINE: No.




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 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.0% for the lysine.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of 3 repetitions

Parameter:

other: %depletion in lysine peptide

Value:

1.38

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:

0

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:

0.69

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 peptide concentration of reference controls A was 0.47 mM for both cysteine and lysine peptides which are equal to 0.50± 0.05 mM.
- Acceptance criteria met for reference controls B and C: yes, the mean peptide concentration of the three Reference Controls C was 0.46 mM and 0.52 mM for cysteine and lysine peptides respectively which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the nine controls B and C was 1.46% and 5.67% for cysteine and lysine peptide which is lower than 15%.
- Acceptance criteria met for positive control: Yes, the depletion mean rate was 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine. The SD of the 3 repetitions was 1.64% (limit < 14.9%) for the percent cysteine depletion and 19.34% (limit <11.6%) for the percent lysine depletion. However, this criterion is considered valid as the third replicate of percent lysine depletion is an outlier based on the results of Grubb’s test and the mean percent lysine peptide depletion of the positive control is 45.60% which is within the range as suggested by OECD 442C test guideline.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 4.78% for cysteine and 8.32% 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: Mean and SD of Percent Peptide depletion for Cysteine and Lysine

For Cysteine peptide
Sample ID	Peak area of cysteine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	972449.0	71.35	71.18	1.64
Cinnamic aldehyde-replicate 2	925851	72.72
Cinnamic aldehyde-replicate 3	1036665	69.46
D027-01 replicate 1	3439003.0	-1.31	-0.64 (Considered as 0)	4.78
D027-01 replicate 2	3243768.0	4.44
D027-01 replicate 3	3565975	-5.05

For Lysine peptide
Sample ID	Peak area of Lysine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	1731217	57.01	45.60	19.34
Cinnamic aldehyde-replicate 2	1750371	56.53
*Cinnamic aldehyde-replicate 3	3089854	23.26
D027-01 replicate 1	4357614	-8.22	1.38	8.32
D027-01 replicate 2	3790534	5.86
D027-01 replicate 3	3765346	6.49

D027-01: 3,3-Dimethyl-8,9-Dinorbornan-2-one

* replicate 3 is an outlier

Table 4: 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	71.18	45.60	58.39	High Reactivity	Positive
D027-01	3,3-Dimethyl-8,9-Dinorbornan-2-one	0	1.38	0.69	No or Minimal Reactivity	Negative

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 cysteine and lysine peptides of 0.69%, 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 test item 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, positive control and reference controls were incubated for 24 hours ± 2 hours at 25°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, and then analyzed with HPLC to measure the peptide depletion. Test samples were prepared in triplicates for both peptides. All validity criteria were fulfilled. The mean of cysteine and lysine peptide depletion by the positive control was 58.39% which shows that it is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of depletion by the test item was 0.69% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, the test item was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).