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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08-08-2019 to 27-08-2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

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
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(1RS,4Z,8E,12RS)-13-oxabicyclo[10.1.0]trideca-4,8-diene
EC Number:
951-761-9
Cas Number:
55722-64-0
Molecular formula:
C12H18O
IUPAC Name:
(1RS,4Z,8E,12RS)-13-oxabicyclo[10.1.0]trideca-4,8-diene
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Other: colourless liquid

In chemico test system

Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- 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) and isopropanol (IPA). At a concentration of 100 mM, ST 10 C 19 was soluble in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), IPA, ethanol and methanol, but not in MQ and ACN:MQ (1:1, v/v). Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study Visual inspection of the forming of a clear solution 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.
- HPLC-PDA (UV) methodology are reported in the full study report.
- Preparation of synthetic peptide solutions [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.7 mg of SPCC in 21.36 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. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.506 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.505 ± 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.
2. Lysine: 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. SPCL Reference Control solutions: 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 SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.505 ± 0.004 mM while the mean peptide concentration of Reference Controls C was 0.515 ± 0.004 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.
- Sample incubations: 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 23 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 1.000 and SPLC r2 = 0.9997)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 71.0% ± 0.6% and SPCL 63.1% ± 0.6%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.6% and SPCL PC : SD = 0.6%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.506 ± 0.002 mM, and 0.505 ± 0.005 mM ; Lysine A, C reference controls: 0.505 ± 0.004 mM and 0.515 ± 0.004 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.3% ; Lysine: Reference Controls B and C was 1.1%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 36.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.82. 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.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile (ACN)
Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Results and discussion

Positive control results:
- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 71.0% ± 0.6% (high reactivity)
- PC LYS-peptide depletion (mean): 63.1% ± 0.6% (high reactivity)

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Mean (%)
Parameter:
other: Cys-peptide depletion
Value:
5.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Negative : No or minimal reactivity
Remarks:
n = 3 ; See 'any other information on results incl. tables' for further information
Key result
Run / experiment:
other: Mean (%)
Parameter:
other: Lys-peptide depletion
Value:
1.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
n = 3 ; Negative: No or minimal reactivity ; See 'any other information on results incl. tables' for further information
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.
- Other: In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (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 Lysine reactivity assay: upon preparation as well as after incubation a phase separation was observed in the CC and the test item samples. In this case, it could not be precise the amount of test item remained in the solution.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

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 variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 1.000 and SPLC r2 = 0.9997)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 71.0% ± 0.6% and SPCL 63.1% ± 0.6%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.6% and SPCL PC : SD = 0.6%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.506 ± 0.002 mM, and 0.505 ± 0.005 mM ; Lysine A, C reference controls: 0.505 ± 0.004 mM and 0.515 ± 0.004 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 0.3% ; Lysine: Reference Controls B and C was 1.1%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 36.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.82. 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.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.

Any other information on results incl. tables

Table 1.0 – Acceptability of the DPRA

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

1.000

>0.99

0.9997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.506 ± 0.002

0.50 ± 0.05

0.505 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.505 ± 0.002

0.50 ± 0.05

0.515 ± 0.004

 

 

 

 

 

CV (%) for RC samples

B and C

<15.0

0.3

<15.0

1.1

Mean peptide depletion PC (cinnamic aldehyde) (%)

60.8-100

71.0

40.2-69.0

63.1

SD of peptide depletion PC (cinnamic aldehyde)

(%)

<14.9

0.6

<11.6

0.6

SD of peptide depletion for the test item

(%)

<14.9

0.6

<11.6

1.2

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

 

Table 2.0 – Results of the DPRA with the test item

SPCC depletion (CYSTEINE)

SPCL depletion (LYSINE)

Mean of SPCC and SPCL depletion

Mean

± SD

Mean

± SD

Test item

5.4%

±0.6%

1.4%

±1.2%

3.4%

 

Applicant's summary and conclusion

Interpretation of results:
other: 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 C&L purposes
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 study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to 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 photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were 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. Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item. 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 phase separation was observed. Since phase separation was observed after the incubation period for SPCL, it cannot be precise the amount of test item which remained in the solution to react with the peptide. In the cysteine reactivity assay the test item showed 5.4% 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 3.4% and as a result the test item was considered to be negative in the DPRA and classified in the “negative or no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met with acceptable restrictions.