[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28-10-2020 until 16-12-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Precipitation and phase separation occurred after the incubation period for both Cysteine and Lysine samples. Phase separation was also observed upon preparation of both samples.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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:

26 June 2020

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:

Reaction mass of (±)-(4RS,4aRS,8RS,8aRS)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE and (±)-(4RS,4aSR,8SR,8aSR)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE and (±)-(4RS,4aSR,8RS,8aSR)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE

Molecular formula:

C14H24O

IUPAC Name:

Reaction mass of (±)-(4RS,4aRS,8RS,8aRS)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE and (±)-(4RS,4aSR,8SR,8aSR)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE and (±)-(4RS,4aSR,8RS,8aSR)-4-ETHYL-8,8a-DIMETHYLOCTAHYDRO-1(2H)-NAPHTHALENONE

Test material form:

liquid

Details on test material:

Physical state: liquid
Storage condition of test material: Dry area, protected from light, in a refrigerator (2-8ºC)
Other: Colourless liquid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
1) Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.2 mg of SPCC in 20.36 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. SPCC Reference Control Solutions: 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.

2) Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 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.

- Preparation of the test chemical solutions:
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e., by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH).
The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer based incubation mixtures. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assays 37.33 mg of test item was pre-weighed into
a clean amber glass vial and dissolved, just before use, in 1792 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before. Any residual volumes were discarded.

- Preparation of the positive controls, reference controls and co-elution controls:
(i) Positive Controls:
Three replicates of Positive Control (PCcys-1 to PCcys-3) were prepared by mixing 750 μL of the Phosphate buffer pH 7.5 solution, 200 μL of the ACN solution and 50 μL of the Cinnamic aldehyde solution (100mM in ACN).
Three replicates of Positive Control (PClys-1 to PClys-3) were prepared by mixing 750 μL of the Stock solution of 0.667 mM SPCL and 250 μL of the Cinnamic aldehyde solution (100mM in ACN).
(ii) Reference controls:
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. The SPCC was subsequently calibrated at multiple concentrations.
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. The SPCL was subsequently calibrated at multiple concentrations.
(iii) Co-elution controls: Co-elution control (CC) solution (CCcys) was prepared by mixing 750 μL of the Phosphate buffer pH 7.5 with 200 μL of the ACN solution and 50 μL of test item test solution (100 mM). Co-elution control (CC) solution (CClys) was prepared by mixing 750 μL of the Ammonium acetate buffer pH 10.2 with 250 μL of the test item test solution (100 mM).

INCUBATION
- Incubation conditions: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the
autosampler and analysis of the first RCcysB- or RClysB-sample was 24.1 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.

- Precipitation noted: Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation and/or phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
Upon preparation of the SPCC and SPCL test item samples, phase separation was observed.
After incubation of the SPCC and SPCL test item samples, both a precipitate and phase
separation was observed.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
1) SPCC calibration curve:
The SPCC standard calibration curve is presented in the full study report. The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
2) SPCL calibration curve:
The SPCL standard calibration curve is presented in the full study report. The correlation coefficient (r2) of the SPCL standard calibration curve was 1.0000. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

- Verification of the suitability of the HPLC for test chemical and control substances:
(i) Acceptability of the Cysteine reactivity assay: The mean peptide concentration of Reference Controls A was 0.499 ± 0.0003 mM and the mean peptide concentration of Reference Controls C was 0.496 ± 0.002 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and
ndicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
(ii) Acceptability of the Lysine reactivity assay:
The mean peptide concentration of Reference Controls A was 0.501 ± 0.001 mM and the mean peptide concentration of Reference Controls C was 0.501 ± 0.003 mM. The means of Reference
Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0.2 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
Precipitation and phase separation occurred after the incubation period for both Cysteine and
Lysine samples. Phase separation was also observed upon preparation of both samples.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for reference controls A to C: All criteria met.
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

Table1: Acceptability of the DPRA

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9998	>0.99	1.0000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.499 ± 0.0003	0.50 ± 0.05	0.501 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.496 ± 0.002	0.50 ± 0.05	0.501 ± 0.003
CV (%) for RC samples B and C	<15.0	0.7	<15.0	0.7
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	71.6	40.2-69.0	62.9
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.2
SD of peptide depletion for the test item (%)	<14.9	0.4	<11.6	0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

 

Table 2: Results of the DPRA with 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	0.2%	±0.4 %	0.0%	±0.0%	0.1%	Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

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 a precipitate and phase separation was observed after the incubation period for both SPCC and SPCL, and phase separation was also observed upon preparation of both samples, one cannot be sure how much test item remained in the solution to react with the peptides. This negative result is uncertain and should be interpreted with due care. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

Conclusions:

The test item gave a negative in DPRA and was classified in the "Negative: No or minimal reactivity class" using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

Executive summary:

The objective of this study was to determine the reactivity of Test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD 442C guideline.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

All the assay validation criteria were met. 

 

The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.

Upon preparation of the SPCC and SPCL test item samples, phase separation was observed. After incubation of the SPCC and SPCL test item samples, both a precipitate and phase separation was observed.

In the cysteine reactivity assay the test item showed 0.2% ±0.4 % SPCC depletion while in the lysine reactivity assay the test item showed 0.0% ±0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.1 % 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.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. 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 a precipitate and phase separation was observed after the incubation period for both SPCC and SPCL, and also phase separation was also observed upon preparation of both samples, one cannot be sure how much test item remained in the solution to react with the peptides. This negative result is uncertain and should be interpreted with due care. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.