1,1,2,3,3,6-hexamethylindan-5-yl methyl ketone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,3,3,6-hexamethylindan-5-yl methyl ketone

EC Number:

239-360-0

EC Name:

1,1,2,3,3,6-hexamethylindan-5-yl methyl ketone

Cas Number:

15323-35-0

Molecular formula:

C17H24O

IUPAC Name:

1,1,2,3,3,6-hexamethylindan-5-yl methyl ketone

Test material form:

solid

Specific details on test material used for the study:

Expiry Date: 09 November 2018
Storage Conditions: at room temperature, protection from light
Description: Off-white solid

In chemico test system

Details on the study design:

PREPARATION OF THE TEST ITEM:
- The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution of 100 mM was prepared.
CONTROLS:
- Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
- Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
- Co-elution Control: Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Reference Control: Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was the same as for the positive control because no (further) solvent was used. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples
TEST SYSTEM
- HPLC System: HPLC/DAD: Agilent, 1200 Series with Chemstation, Rev. B.04.01; Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution; Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm, Agilent Art. Nr. 861753-902; Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm; Column Temperature: 30°C; Sample Temperature: 25°C; Run Time: 20 minutes; Gradient: Time (0 min, 10 min, 11 min, 13 min, 13.5 min), Flow: 0.35 mL/min (all time points), %A: 90, 75, 10, 10, 90, %B: 10, 25, 90, 90, 10, End run at 20 min. Injection Volume: 10µL
- HPLC Mobile Phase A: 0.1% v/v trifluoroacetic acid in water; HPLC Mobile Phase B: 0.085% v/v trifluoroacetic acid in acetonitrile
- Peptides: 20.02 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.93 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 19.82 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.63 mL) to reach a concentration of 0.667 mM. All peptides used for this study were stored at -80°C and protected from light. Peptides were thawed only immediately prior to use.
PRE-EXPERIMENTS:
- Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvent suitable for the test: acetonitrile
EXPERIMENTAL PROCEDURE
- Incubation of the Test Item with the Cysteine and Lysine Peptide: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5°C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.
- Preparation of the HPLC Standard Calibration Curve: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/ v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range 0-0.534 mM.
- HPLC Preparation and Analysis: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30°C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.
DATA ANALYSIS
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula: PPD = (1-(Peptide Peak Area in the Replicate Injection/Mean Peptide Peak Area in Reference Control C))*100. Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1" if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS "No Category", if the mean depletion of both peptides is below 6.38%. In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model).
ACCEPTANCE CRITERIA:
The run meets the acceptance criteria if: the standard calibration curve has a r2 > 0.99, the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%, the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 1 1.6%, the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM, the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if: the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD), the maximum standard deviation (SD) for the test chemical replicates is < 1 1.6% for the lysine percent depletion (PPD), the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.21%.

In vitro / in chemico

Results

Key result

Parameter:

mean cystein depletion

Value:

68 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

- Pre-Experiments: Solubility of the test item was determined prior to the main experiment. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.
- Precipitation and Phase Separation: All test item solutions were freshly prepared immediately prior to use. For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.
- Co-elution with the Peptide Peaks: No co-elution of the test item with any of the peptide peaks was observed.
- The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was > 0.99 (0.9992). The mean peptide depletion of the positive control was between 60.8% and 100% (69.31%). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5119 mM, RC C acetonitrile. 0.5079 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.48%, RC C acetonitrile: 0.30%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 14.9% (PC: 0.45%; test item: 0.18%). For the lysine run the coefficient of determination for the calibration curve was > 0.99 (09999). The mean peptide depletion of the positive control was between 40.2% and 69.0% (63.12%). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5008 mM, RC C acetonitrile 0.4926 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 1.57%, RC C acetonitrile. 1.19%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 11.6% (PC: 1.08%; test item: 0.74%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met