Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-04-16 to 2019-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate

EC Number:

949-569-5

Molecular formula:

C12H22O2

IUPAC Name:

Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate

Test material form:

liquid

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)

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:
22.52 mg and 31.47 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1136 μL and 1587 μL ACN, respectively, to obtain 100 mM solutions. No correc tion 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 11.0 mg of SPCC in 21.96 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 10.4 mg of SPCL in 20.08 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 26 hours for RCcysB-samples and 25 hours for the RClysBsamples. All samples were analysed with HPLC analysis. Prior to HPLC analysis the samples were visually inspected for precipitation. The samples that showed precipitation/phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean % SPCC depletion

Value:

1.4 %

Positive controls validity:

valid

Remarks:

80 ± 0.1 %

Results 2

Key result

Parameter:

other: mean % SPCL depletion

Value:

0.1 %

Positive controls validity:

valid

Remarks:

58.8 ± 0.7 %

Results 3

Key result

Parameter:

other: mean % depletion

Value:

0.8 %

Positive controls validity:

valid

Any other information on results incl. tables

Upon preparation as well as after incubation of the SPCC test item samples a precipitate was observed. Upon preparation of the SPCL test item samples a phase separation was observed while after incubation of these samples a precipitate was observed.

In the cysteine reactivity assay the test item showed 1.4 % SPCC depletion while in the lysine reactivity assay the test item showed 0.1 % SPCL depletion. The mean of the SPCC and SPCL depletion was 0.8 % and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

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
Test item	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
	1.4 %	± 0.4 %	0.1 %	± 0.2 %	0.8 %	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	417946	0.100	79.9 %
PCcys-2	414846	0.099	80.0 %
PCcys-3	413080	0.099	80.1 %
		Mean	80.0 %
		SD	0.1 %

SD = Standard Deviation

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)
210251/A-cys-1	2056772	0.498	1.0 %	54723	37.59
210251/A-cys-2	2043642	0.495	1.6 %	54528	37.48
210251/A-cys-3	2041676	0.494	1.7 %	54442	37.50
		Mean	1.4 %	NA	37.52
		SD	0.4 %	NA	0.06

SD = Standard Deviation, 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)	SPCC Depletion
PClys-1	666448	0.203	59.5 %
PClys-2	674337	0.206	59.0 %
PClys-3	689184	0.210	58.1 %
		Mean	58.8 %
		SD	0.7 %

SD = Standard Deviation

Table 5. SPCL 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)
210251/A-lys-1	1654675	0.507	0.0 %	51344	32.23
210251/A-lys-2	1647770	0.505	0.0 %	53515	30.79
210251/A-lys-3	1647770	0.502	0.3 %	52392	31.28
		Mean	0.1 %	NA	31.43
		SD	0.2 %	NA	0.73

SD = Standard Deviation, NA = Not Applicable.

 

Applicant's summary and conclusion

Interpretation of results:

other: negative for dendritic cells activation

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 after the incubation period for both SPCC and
SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

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 26 and 25 hours, respectively, at 25 °C, the relative peptide concentration was determined by HPLC detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 100 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-RFAAKAACOOH) were prepared in ACN. Co-eluent samples containing 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 HPLC 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 1.4 % and the mean % SPCL depletion was 0.1 %. The average % depletion of the test item was determined to be 0.8 %. The reference control showed mean % SPCC depletion of 80 % and a mean % SPCL depletion of 58.8 %. All validity criteria were met and the test is considered as valid. 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 after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.