Cyclopentene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2018 - 24 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours of incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at
220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

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 (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Of the three human skin sensitisation tests that reflect Key Events in the Adverse Outcome Pathway (AOP), the Direct Peptide Reactivity Assay (DPRA) assay represents Key Event 1, Covalent binding of the electrophilic substance to proteins. Test materials are classified based on the results of “two out of three” tests.
The the DPRA assay is fully accepted at a regulatory level for the hazard identification of Skin Sensitisers and Non-Sensitisers in accordance with the UN GHS. This is fully approved by the EU Cosmetics Regulation 1223/2009, CLP Regulation 1272/2008 and REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) legislation.

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentene

EC Number:

205-532-9

EC Name:

Cyclopentene

Cas Number:

142-29-0

Molecular formula:

C5H8

IUPAC Name:

cyclopentene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Clear colourless liquid
- Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
- Batch No.of test material: 0000015386
- Expiration date of the batch: 19 March 2021
- Purity: 99.28%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In redrigerator (2-8°C)
- Stability under storage conditions: Yes until 30 November 2021

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Solubility of the test material in an appropriate solvent was assessed before the DPRA was performed. Acetonitrile (ACN), Milli-Q water (MQ); ACN:MQ (1:1, v/v); isopropanol, acetone: ACN (1:1 v/v) and dimethylsulphoxide (DMSO):ACN (1:9, v/v) were evaluated.
- Test material stock solutions were prepared freshly for each reactivity assay.
- 13.30 mg test material was pre-weighed into a clean amber glass vial and dissolved in 1953 μL ACN prior to use to obtain a 100 mM solution. Visual inspection of a clear soltion was considered sufficient to confirm that the test material was dissolved.
- Preliminary purification step (if any): No correction for the purity or composition of the test item.
- Final dilution of a dissolved solid, stock liquid or gel: 100 mM solution (cyclopentene in ACN)
- Samples of the test material, positive controls and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay. Any residual volumes were discarded.

In chemico test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

TEST SYSTEM
- Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight for SPCC was 750.9 g/mol and for SPCL it was 775.9 g/mol.

CYSTEINE REACTIVITY ASSAY
> Preparation of solutions: 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 mixutre was stirred for 5 minutes and then sonicated for 5 more minutes.
> SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcys B, RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
> SPCC Calibration curve: please refer to Table 1.
> The co-elution (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as seen in Table 2.

LYSINE REACTIVITY ASSAY
> Preparation of solutions: 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 then stirred for 5 minutes.
> SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB, RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
> SPCL Calibration Curve: please refer to Table 3.
> The co-elution (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as seen in Table 4.

SAMPLE INCUBATIONS
- After preparation, all samples (including reference, control and calibration samples) were placed in the autosampler in the dark and incubated at 25±2.5°C.
- The incubation time from the placing the samples in the autosampler and the analysis of the first RCcysB- or RClysB-sample was 25 hours.
- The time between the first RCcysB- or RClysB-injectin and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
- Samples were visually inspected for precipitation before HPLC-PDA analysis.

HPLC-PDA Analysis
- SPCC and SPCL peak areas in the samples were measured by HPLC-PDA.
- Sample analysis was performed according to the the systems show in Figure 1.

Results and discussion

Positive control results:

CYSTEINE ASSAY: The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 68.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

LYSINE ASSAY: The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was57.3% ± 3.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Mean ± SD

Parameter:

other: SPCC Depletion

Value:

3.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± SD 1.7%

Remarks:

Negative DPRA Prediction: No or minimal reactivity

Results 2

Key result

Run / experiment:

other: Mean ± SD

Parameter:

other: SPCL Depletion

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± SD 2.5%

Remarks:

Negative DPRA Prediction: No or minimal reactivity

Results 3

Key result

Run / experiment:

mean

Parameter:

other: Mean of SPCC and SPCL depletion

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative DPRA depletion

Remarks:

No or minimal reactivity

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Yes

Any other information on results incl. tables

SOLUBILITY ASSESSMENT OF TEST MATERIAL

- At a concentration of 100 mM, cyclopentene was not soluble in MQ, ACN:MQ (1:1 v/v), acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v), but was soluble in ACN and isopropanol.

- ACN is the preferred solvent for DPRA and so was used to dissolve the test material in this study.

ACCEPTABILITY OF CYSTEINE REACTIVITY ASSAY

- The correlation coefficient (r-squared) of the SPCC standard calibration curve was 0.997. The standard calibration curve was accepted since the r-squared value was > 0.99.

- The mean peptide concentrations of Reference Controls A (0.510 ±0.07 mM) and Reference Controls C (0.499 ±0.007 mM) were both within the acceptance range of (0.50 ±0.05 mM), confirming the suitability of the HPLC system and indicating that the ACN solvent did not affect the Percent SPCC Depletion.

- The Coefficient of Variation (CV) of the peptide areas for the 9 Reference Controls B and C was 2.3%, which was within the acceptance criteria (CV < 15%) and thus confirms the stability of the HPLC run over time.

- The mean area ratio (A220/A258) of the Reference Control samples was 18.12. The mean A220/A258 ratio ± 10% range was 16.31-19.94. Each sample showed an A220/A258 ratio within this range, signifying that co-elution has not occurred.

> RESULTS

- Preparation of a 100mM cyclopentene stock solution in ACN showed that the test material dissolved completely.

- Upon preparation, a phase separation was observed in the co-elution control (CC) and test material samples, but was not seen after incubation of the test material samples.

- In the CC sample, there was no peak observed at the retention time of SPCC, which showed that there was no co-elution of the test material with SPCC.

- For the 209393/A-cys samples, the mean SPCC A220/A258 area ratio was 18.35, which was within the 16.31-19.94 range, further corroborating that there was no co-elution.

- The mean Percent SPCC Depletion for the test item was 3.1% ± 1.7%.

ACCEPTABILITY OF CYSTEINE REACTIVITY ASSAY

- The correlation coefficient (r-squared) of the SPC; standard calibration curve was 0.991. The standard calibration curve was accepted since the r-squared value was > 0.99.

- The mean peptide concentrations of Reference Controls A (0.510 ±0.020 mM) and Reference Controls c (0.520 ±0.003 mM) were both within the acceptance range of (0.50 ±0.05 mM), confirming the suitability of the HPLC system and indicating that the ACN solvent did not affect the Percent SPCC Depletion.

- The Coefficient of Variation (CV) of the peptide areas for the 9 Reference Controls B and C was 3.3%, which was within the acceptance criteria (CV < 15%) and thus confirms the stability of the HPLC run over time.

- The mean area ratio (A220/A258) of the Reference Control samples was 16.14. The mean A220/A258 ratio ± 10% range was 14.52-17.75. Each sample showed an A220/A258 ratio within this range, signifying that co-elution has not occurred

> RESULTS

- Preparation of a 100mM cyclopentene stock solution in ACN showed that the test material dissolved completely.

- Upon preparation, a phase separation was observed in the co-elution control (CC) and test material samples, but was not seen after incubation of the test material samples.

- In the CC sample, there was no peak observed at the retention time of SPCC, which showed that there was no co-elution of the test material with SPCC.

- For the 209393/A-cys samples, the mean SPCC A220/A258 area ratio was 16.32, which was within the 14.52 -17.75 range, further corroborating that there was no co-elution.

- The mean Percent SPCC Depletion for the test item was 1.4% ± 2.5%.

Table. 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
Cyclopentene	3.1%	±1.7%	1.4%	±2.5%	2.3%	Negative: No or minimal reactivity

SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Cyclopentene was found to be negative in the DPRA assay. In the cysteine reactivity assay the test material showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% 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.

Executive summary:

The aim of this study was to determine the reactivity of the test material, Cyclopentene, towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL), according OECD 442C guideline.

The test material was dissolved in acetonitrile to obtain a final 100 mM solution. The solubility of the test material in several solvents was investigated prior to conducting the DPRA assay. Following incubation of the test material with either SPCC or SPCL, the relative peptide cocnentration was determined by HPLC with gradient elution and photodiode array (PDa) detection at 220 nm and 258nm. SPCC and SPCL percent depletion values were calculated and used in the Cysteine 1:10/Lysine 1:50 prediction model, which allows the test material to be assigned to one of four reactivity classes in order to be able to discriminate between sensitisers and non-sensitisers.

The validation parameters were all within the acceptability criteria for the DPRA. A phase separation was observed in the SPCC and SPCL test material samples upon preparation, but disappeared following incubation.

In conclusion, this DPRA test was considered to be valid. Cyclopentene was found to be negative in the DPRA assay. In the cysteine reactivity assay the test material showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% 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.