13-ethyl-11-methylene-gon-4-ene-3,17-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 October 2020 - 6 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Cas Number:

54024-17-8

Molecular formula:

C20 H26 O2

IUPAC Name:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Test material form:

solid: particulate/powder

Details on test material:

Storage Conditions: In refrigerator (2-8°C)

Specific details on test material used for the study:

Physical Description: Off-white powder
Storage conditions: In refrigerator (2-8°C)
Test item handling: No specific handling conditions required

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:
SPCC: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.1 mg of SPCC in 20.16 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 10.3 mg of SPCL in 19.88 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: No correction for the purity/composition of the test item was performed.
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), and 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 assay 55.92 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1874 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 starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the positive controls, reference controls and co-elution controls

INCUBATION
- Incubation conditions: incubated at 25 ± 2.5°C in the dark for 23.7 hours.

PREPARATION OF THE HPLC
All samples were analyzed according to the HPLC-PDA method presented in Table 1. (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2. (See 'other information on materials and methods').

DATA EVALUATION
- The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-((Peptide Peak Area in Replicate Injection (at 220 nm))/(Mean Peptide Peak Area in Reference Controls (at 220 nm)))]×100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% mean area ratio of control samples<110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION
See table 3 (See 'other information on materials and methods').

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Parameter:

lysine depletion

Value:

3.7 %

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Parameter:

cysteine depletion

Value:

2.4 %

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Acceptability of the Cysteine Reactivity Assay:
- The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9994. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.500 ± 0.001 mM and the mean peptide concentration of Reference Controls C was 0.499 ± 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 indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C
was 1.0%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 37.85. The mean A220/A258 ratio ± 10% range was 34.06-41.63. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.2% ± 1.6%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item
Preparation of a 100 mM EMETAM stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 210394/A-cys samples, the mean SPCC A220/A258 area ratio was 37.77. Since this was within the 34.06-41.63 range, this again indicated that there was no co-elution of the test item with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 2.4% ± 0.6%.

Acceptability of the Lysine Reactivity Assay
- The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9994. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.519 ± 0.003 mM and the mean peptide concentration of Reference Controls C was 0.515 ± 0.012 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.
- The CV of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 30.71. The mean A220/A258 ratio ± 10% range was 27.64-33.79. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 63.2% ± 2.0%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item
Preparation of a 100 mM EMETAM stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.
In the CC
sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 210394/A-lys samples, the mean SPCL A220/A258 area ratio was 30.31. Since this was within the 27.64-33.79 range, this again indicated that there was no co-elution of the test item with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the test item was 3.7% ± 0.4%.

Any other information on results incl. tables

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

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
EMETAM	2.4%	±0.6%	3.7%	±0.4%	3.1%	Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

A DPRA assay was performed according to OECD 442C and in accordance with GLP principles. In conclusion, this DPRA test is valid. EMETAM 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 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. The percentages of SPCL depletion might be underestimated.

Executive summary:

A DPRA assay was performe according to OECD 442C and in accordance with GLP principles. The objective of this study was to determine the reactivity of EMETAM 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.

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.

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 as well as after incubation of the SPCC test item samples, no precipitate or phase separation was observed in any of the samples. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 2.4% SPCC depletion while in the lysine reactivity assay the test item showed 3.7% SPCL depletion. The mean of the SPCC and SPCL depletion was 3.1% 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. EMETAM 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 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. The percentages of SPCL depletion might be underestimated.