cis-2-tert-butylcyclohexan-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 November 2021 to 16 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

14 June 2021

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

12 January 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-tert-butylcyclohexan-1-ol

EC Number:

230-601-5

EC Name:

cis-2-tert-butylcyclohexan-1-ol

Cas Number:

7214-18-8

Molecular formula:

C10H20O

IUPAC Name:

cis-2-tert-butylcyclohexan-1-ol

Test material form:

solid

In chemico test system

Details of test system:

other:

Details on the study design:

Preparation of the Test Item: The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. 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 with a concentration of 100 mM was prepared. A factor of 1.14 was used to correct for the purity of the test item.

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 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.

Peptides: 20.42 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (40 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 20.66 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 (39.88 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.

Dose Groups:
- Reference Control C (solvent control)
- Test Item: 100 mM stock solution
- Positive Control: 100 mM stock solution

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 solvents suitable for the test: acetonitrile
- The test item was completely soluble in acetonitrile, therefore, acetonitrile was chosen as suitable vehicle for the main experiments.

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.

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

The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.

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 < 11.6%,
- 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 < 11.6% 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.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

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 68.50%.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.93 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Precipitation and Phase Separation: For the 100 mM stock 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. No precipitation, turbidity or phase separation was observed for any of the samples.  For the 100 mM stock solution of the test item turbidity 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 test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Co-elution with the Peptide Peaks : No co-elution of the test item with any of the peptide peaks was observed.

To detect a co-elution of the test item with the peptide peak, a ratio of the 220 nm peak area and the 258 nm peak area was calculated too (peak purity indication). If the ratio of the control samples and the test item samples do not differ more than 10% from each other, no sign for a co-elution is given.

For the cysteine measurement the peak purity indication was < 10% (0.5 % test item replicate 1; 0.4 % test item replicate 2; 0.7 % test item replicate 3; mean: 0.3 %) and for the lysine measurement the peak purity indication was < 10% (0.3 % test item replicate 1; 0.8 % test item replicate 2; 0.3 % test item replicate 3; mean: 0.1 %).

Cysteine and Lysine Values of the Calibration Curve:

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	16.1350	0.5340	14.0090	0.5340
STD2	8.1080	0.2670	6.8920	0.2670
STD3	4.0320	0.1335	3.4080	0.1335
STD4	1.9610	0.0667	1.7000	0.0667
STD5	0.9660	0.0334	0.8720	0.0334
STD6	0.4700	0.0167	0.4500	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.1070	0.1364	72.76	72.98	0.20	0.27
	4.0500	0.1345	73.13
	4.0630	0.1350	73.05
Test Item	15.3380	0.5071	0.00	0.93	0.93	99.76
	14.9340	0.4937	0.94
	14.7950	0.4891	1.86

Depletion of the Lysine Peptide:

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.6070	0.1770	63.94	64.03	0.07	0.11
	4.5890	0.1763	64.09
	4.5940	0.1765	64.05
Test Item	12.8560	0.4919	0.00	0.00	0.00	n/a
	12.8720	0.4925	0.00
	12.8620	0.4921	0.00

 

Categorization of the Test Item:

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	--	--	--	0.93	Minimal Reactivity	negative
Positive Control	68.50	High Reactivity	positive	72.98	Moderate Reactivity	positive

 

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide.

Executive summary:

The study was conducted to determine the sensitising potential of Verdol according to the OECD Guideline 422C in compliance with GLP. Based on a molecular weight of 156.26 g/mol a 100 mM stock solution was prepared in acetonitrile. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock 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. No precipitation, turbidity or phase separation was observed for any of the samples. For the 100 mM stock solution of the test item turbidity 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 test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Phase separation in the lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.93%). 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 68.50%. The controls confirmed the validity of the study for both, the cysteine and lysine run.

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide.