Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-01-03 to 2018-02-27

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:

Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

EC Number:

947-711-0

Molecular formula:

C14H28O

IUPAC Name:

Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptides:
Cysteine- (SPCC) containing peptide: Ac-RFAACAA-COOH (MW=750.9 g/mol)
Lysine- (SPCL) containing peptide: Ac-RFAAKAA-COOH (MW=775.9 g/mol)

Material and technical equipment:
- System 1 (used for Cysteine Reactivity Assay)
Surveyor MS HPLC pump
MPS 3C autosampler
LC Column oven 300
Surveyor PDA detector
- System 2 (used for Lysine Reactivity Assay)
Surveyor MS HPLC pump
HTC PAL autosampler
Column Oven #151006
Surveyor PDA detector

Controls:
For Cysteine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, SPCC stock solution and ACN
Co-elution control: Phosphate buffer pH 7.5, ACN and the test substance without peptide.
For Lysine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, and SPCL stock solution
Co-elution control: Ammonium acetate buffer pH 10.2 and the test substance without peptide.

Test substance preparation:
33.21 mg and 35.25 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1563 µL and 1659 µL ACN, respectively, to obtain 78.2 mM solutions. No correction for the purity/composition of the test item was performed.
Vehicle: Acetonitrile, ACN
Reason for the vehicle: A solubility test to assesse the test items solubility in different solvents was performed before the main test. ACN was chosed as the best solvent in which the test item dissolved and did not for precipitation or cloudy solution.

Test Item Samples:
- Test item solution, ACN and SPCC stock solution
- Test item and SPCL stock solution

Preparation of peptide stock solutions:
SPCC - A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
SPCL - A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes. Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

Experimental procedure:
After preparation, the samples were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation period was 24.5 hours for RCcysB-samples and 23 hours for the RClysB-samples. All samples were analysed with HPLC-PDA analysis. Prior to HPLC PDA analysis the samples were visually inspected for precipitation.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean % SPCC depletion

Value:

0.46

Positive controls validity:

valid

Remarks:

78.3 %

Results 2

Key result

Parameter:

other: mean % SPCL depletion

Value:

0

Positive controls validity:

valid

Remarks:

52.8 %

Results 3

Key result

Parameter:

other: mean % depletion

Value:

0.23

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
Formation of precipitation of the test item by addition of peptide solution.

Any other information on results incl. tables

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

In the cysteine reactivity assay the test item showed 0.46 % SPCC depletion while in the lysine reactivity assay the test item showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 0.23 % and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test item was considered to be negative in the DPRA.

Since no correction was used to prepare the test item stock solution in ACN, the prepared test item stock solution had a concentration of 78.2 mM instead of the desired 100 mM and the SPCC and SPCL incubations were performed at lower concentrations than intended. However, since at this lower concentration precipitation was already observed upon addition of the test item to the SPCC and SPCL peptide solutions, not applying a correction factor to correct for the purity (78.2 %) of the test item had no impact on the outcome of the study.

Table 1. SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test item	0.46 %	±0.43 %	0.0 %	±0.0 %	0.23 %	Negative: No or minimal reactivity

SD = Standard Deviation

Table 2. SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCC Depletion
PCcys-1	567923	0.112	77.0 %
PCcys-2	568110	0.112	77.0 %
PCcys-3	476032	0.093	80.7 %
		Mean	78.3 %
		SD	2.1 %

Table 3. SPCC Peak Area, Concentration, Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Test Item Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCC Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209239/A-cys-1	2450576	0.502	0.8 %	124186	19.73
209239/A-cys-2	2457863	0.504	0.5 %	126457	19.44
209239/A-cys-3	2483781	0.509	0.0 %	128952	19.26
		Mean	0.46 %	NA	19.48
		SD	0.43%	NA	0.24

NA = Not applicable

Table 4. SPCL Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCL Depletion
PClys-1	1108440	0.234	51.1 %
PClys-2	1055792	0.222	53.4 %
PClys-3	1049112	0.221	53.7 %
		Mean	52.8 %
		SD	1.4 %

Table 5. SPCL Peak Area, Concentration and Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Test Item Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCL Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209239/A-lys-1	2347019	0.513	0.0 %	146582	16.01
209239/A-lys-2	2277900	0.497	0.0 %	145013	15.71
209239/A-lys-3	2302332	0.503	0.0 %	146599	15.70
		Mean	0.0 %	NA	15.81
		SD	0.0 %	NA	0.18

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive for dendritic cells activation (due to precipitation)

Conclusions:

In conclusion, the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, the result is regarded as 'inconclusive for dendritic cells activation (due to precipitation)'.

Executive summary:

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with SPCC or SPCL for 24 hours at 25 °C, the relative peptide concentration was determined by HPLC-PDA detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 78.2 mM and the reference item at 100 mM. As a vehicle acetonitrile (ACN) was used. Synthetic peptide control solutions of SPCC (containing Ac-RFAACAA-COOH) and SPCL (containing Ac-RFAAKAA-COOH) were prepared in ACN. Co-eluent samples containig only the test item and the respective buffer were also tested under the same conditions. After the incubation period samples were injected directly into the HPL-PDA system. The concentration of SPCC or SPCL was photometrically determined at 220 and 258 nm. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. Following this model, the mean % SPCC depletion of the test item was 0.46 % and the mean % SPCL depletion was 0 %. The average % depletion of the test item was determined to be 0.23 %. The reference control showed mean % SPCC depletion of 78.3 % and a mean % SPCL depletion of 52.8 %. All validity criteria were met and the test is considered as valid. As a precipitation was observed after adding the peptide solutions to the test item, the result is regarded as 'inconclusive for dendritic cells activation (due to precipitation)'.