Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-08/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
D-Ribose is a naturally occuring monsaccharyde essential for energy production in mitochondria.
As D-Ribose is essential for life in eukaryotes, sensitization would be lethal, and no organism targeted by sensitization hazard assessment is possible to response positively to sensitization challenge. Therefore, LLNA is not relevant for D-Ribose.
The present DPRA assay confirms the absence of sensitizing potential of Riboxyl, i.e. D-Ribose with minor amount of impureties, as the molecular initiating event of the sensitization AOP cannot be reproduced in chemico.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Dry powder, white to slightly yellow
Specific details on test material used for the study:
Test Item (Riboxyl TM)
Identification:
Riboxyl TM
Appearance:
White to slightly yellow powder

Purity/Composition:
See Certificate of Analysis
Test item storage:
At room temperature protected from light

Additional information
Test Facility test item number:
209457/A
Purity/Composition correction factor:
No correction factor required
Chemical name (IUPAC, synonym or trade name:
A-D(-) ribofuranose; D-ribose
CAS number:
50-69-1

In chemico test system

Details on study design:
Dose Formulation and Analysis
4.5.1. Preparation of Test Item
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) and Milli-Q water (MQ).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 28.93 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1927 μL MQ after vortex mixing to obtain a 100 mM solution. 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.
Any residual volumes were discarded.
4.6. Test System
Test system
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Rationale
Recommended test system in the international OECD guideline for DPRA studies.
Final Report Page 13
Test Facility Study No. 20152806
Source
JPT Peptide Technologies GmbH, Berlin, Germany.
Batch
See Appendix 7 for detailed information.
Storage
The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.
4.7. Reagents
Acetonitrile (ACN)
HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate
Fractopur, Merck, Darmstadt, Germany
Ammonium hydroxide
25%, Merck
Disodium hydrogen phosphate (Na2HPO4·12H2O)
Emsure, Merck
MilliQ-water (MQ)
Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate (NaH2PO4·H2O)
Emsure, Merck
Trifluoroacetic acid (TFA)
>99%, Sigma Aldrich, Zwijndrecht, The Netherlands
4.8. Experimental Design
4.8.1. Preparation of Solutions for Cysteine Reactivity Assay
4.8.1.1. Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
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 mixture was stirred for 5 minutes followed by 5 minutes sonication.
4.8.1.2. 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. In addition, a RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL MQ.
4.8.1.3. SPCC Calibration Curve
A SPCC calibration curve was prepared as described in the table below.

4.8.2. Preparation of Solutions for Lysine Reactivity Assay
4.8.2.1. Synthetic Peptide Containing Lysine (SPCL) Stock Solution
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 followed by stirring for 5 minutes.
4.8.2.2. 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. In addition, a RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL MQ.
4.8.2.3. SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in the table below.
4.8.3. 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 24 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.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
4.8.4. HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)
Final Report Page 16
Test Facility Study No. 20152806
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).
5. ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.
6. ANALYSIS
6.1. Data Evaluation
The concentration of SPCC or SPCL was photometrically 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%6.2. Data Interpretation
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

Positive control results:
The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). 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 50.2% ± 4.0%. 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
Key result
Parameter:
other: SPCC/SPCL depletion (%)
Value:
ca. 2.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
9.1. Solubility Assessment of the Test Item
At a concentration of 100 mM, Riboxyl TM was not soluble in ACN, but was soluble in MQ. Therefore this solvent was used to dissolve the test item in this DPRA study.
9.2. Cysteine Reactivity Assay
The reactivity of Riboxyl TM towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209457/A and 209457/A- cys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).
9.2.1. Acceptability of the Cysteine Reactivity Assay
The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.996. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The results of the Reference Control samples A, C and CMQ are presented in Table 4 (Appendix 3). The mean peptide concentration of Reference Controls A was
0.524 ± 0.001 mM, the mean peptide concentration of Reference Controls C was
0.529 ± 0.004 mM and the mean peptide concentration of Reference Controls CMQ was
0.508 ± 0.008 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCC Depletion.
The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.5%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 18.28. The mean A220/A258 ratio ± 10% range was 16.45-20.11. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). 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 70.3% ± 1.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
9.2.2. Results Cysteine Reactivity Assay for the Test Item
Preparation of a 100 mM Riboxyl TM stock solution in MQ showed that the test item was dissolved completely. 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.
The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 209457/A-cys samples, the mean SPCC A220/A258 area ratio was

18.10. Since this was within the 16.45-20.11 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 CMQ. The mean Percent SPCC Depletion for the test item was 0.3% ± 0.6%.
9.3. Lysine Reactivity Assay
The reactivity of Riboxyl TM towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 24 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209457/A and 209457/A- lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).
9.3.1. Acceptability of the Lysine Reactivity Assay
The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.992. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The results of the Reference Control samples A, C and CMQ are presented in Table 10 (Appendix 3). The mean peptide concentration of Reference Controls A was
0.527 ± 0.011 mM, the mean peptide concentration of Reference Controls C was
0.477 ± 0.008 mM and the mean peptide concentration of Reference Controls CMQ was
0.469 ± 0.005 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCL Depletion.
The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 5.0%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 16.03. The mean A220/A258 ratio ± 10% range was 14.43-17.64. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). 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 50.2% ± 4.0%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
9.3.2. Results Lysine Reactivity Assay for the Test Item
Preparation of a 100 mM Riboxyl TM stock solution in MQ 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.
The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the 209457/A-lys samples, the mean SPCL A220/A258 area ratio was

15.77. Since this was within the 14.43-17.64 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 CMQ. The mean Percent SPCL Depletion for the Test Item was 4.8% ± 1.4%.
9.4. DPRA Prediction and Reactivity Classification
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
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 0.3% SPCC depletion while in the lysine reactivity assay the test item showed 4.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.6% 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.

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

RiboxylTM

0.3%

±0.6%

4.8%

±1.4%

2.6%

Negative: No or minimal

reactivity

SD = Standard Deviation.

Table 1

HPLC-PDA Method for Determination of SPCC and SPCL

 

Mobile phase

A: 0.1% (v/v) TFA in Milli-Q water

B: 0.085% (v/v) TFA in ACN

Gradient

Cysteine

Lysine

0min             10% B

10  min           25% B

11  min           90% B

13 min           90% B

13.5min         10% B

20 min           10% B

0min             10% B

10  min           20% B

11  min           90% B

13 min           90% B

13.5min        10% B

20 min           10% B

Flow

0.35 mL/min

Injection volume

3 µL

Sample tray temperature

Set at 25°C

Column

Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 µm(Agilent Technologies, Santa Clara, CA, USA)

Guard column

SecurityGuard™ cartridge for C18, 4 x 2.0 mm(Phenomenex, Torrance, CA, USA)

Column temperature

Set at 30°C

Detection

Photodiode array detection, monitoring at 220 and 258 nm

Table 2

Analysis Sequences of Cysteine and Lysine Reactivity Assays

 

Sample description

Cysteine reactivity assay

Lysine reactivity assay

Calibration standards (STD) and reference controls (RC)

STDcys7 STDcys6 STDcys5 STDcys4 STDcys3 STDcys2 STDcys1 DilutionbufferRCcysA-1

RCcysA-2 RCcysA-3

STDlys7 STDlys6 STDlys5 STDlys4 STDlys3 STDlys2 STDlys1 DilutionbufferRClysA-1

RClysA-2 RClysA-3

Co-elution controls (CC)

CCcys-209457/A

CClys-209457/A

Reference controls (RC)

RCcysB-1 RCcysB-2

RCcysB-3

RClysB-1 RClysB-2

RClysB-3

First set of replicates

RCcysC-1RCcysCMQ-1PCcys-1

209457/A-cys-1

RClysC-1RClysCMQ-1PClys-1

209457/A-lys-1

Second set of replicates

RCcysC-2RCcysCMQ-2PCcys-2

209457/A-cys-2

RClysC-2RClysCMQ-2PClys-2

209457/A-lys-2

Third set of replicates

RCcysC-3RCcysCMQ-3PCcys-3

209457/A-cys-3

RClysC-3RClysCMQ-3PClys-3

209457/A-lys-3

Reference controls

RCcysB-4 RCcysB-5

RCcysB-6

RClysB-4 RClysB-5

RClysB-6

STD = standard; RC = reference control; CC = co-elution control; PC = positive control.

Table 3

SPCC Retention Times and Peak Areas (at 220 nm and 258 nm)

 

Sample code

Retention time of peak

at 220 nm (min)

Peak area at 220 nm

(µAU)

Peak area at 258 nm

(µAU)

STDcys7

NF

NF

NF

STDcys6

7.87

117400

4779

STDcys5

7.63

200865

8773

STDcys4

7.58

414923

17859

STDcys3

7.65

703504

36375

STDcys2

7.58

1382125

70419

STDcys1

7.48

2491502

141041

Dilution buffer

NF

NF

NF

RCcysA-1

7.52

2499196

136437

RCcysA-2

7.73

2489862

133983

RCcysA-3

7.55

2497917

137622

CCcys-209457/A

NF

NF

NF

RCcysB-1

7.68

2460974

137687

RCcysB-2

7.65

2456420

139124

RCcysB-3

7.58

2485406

138536

RCcysC-1

7.50

2537224

136421

RCcysCMQ-1

7.63

2443216

145506

PCcys-1

7.63

712190

33881

209457/A-cys-1

7.60

2444865

134492

RCcysC-2

7.53

2502182

136318

RCcysCMQ-2

7.47

2447575

133831

PCcys-2

7.88

778302

35549

209457/A-cys-2

7.80

2399294

135057

RCcysC-3

7.53

2514301

135985

RCcysCMQ-3

7.57

2378231

132453

PCcys-3

7.53

753856

38680

209457/A-cys-3

7.83

2439721

133001

RCcysB-4

7.65

2415990

132903

RCcysB-5

7.53

2498480

132808

RCcysB-6

7.60

2442610

132718

NF = No peak found.


Table 4

SPCC Peak Area at 220 nm and Concentration in Reference Controls A, C and CMQ

 

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RCcysA-1

2499196

0.525

RCcysA-2

2489862

0.523

RCcysA-3

2497917

0.524

 

Mean

0.524

SD

0.001

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RCcysC-1

2537224

0.533

RCcysC-2

2502182

0.525

RCcysC-3

2514301

0.528

 

Mean

0.529

SD

0.004

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RCcysCMQ-1

2443216

0.512

RCcysCMQ-2

2447575

0.513

RCcysCMQ-3

2378231

0.498

 

Mean

0.508

SD

0.008

 


SD = Standard Deviation.


Table 5


SPCC Peak Area of Reference Controls B and C

 

Reference Controls in ACN

Peak area at 220 nm (µAU)

RCcysB-1

2460974

RCcysB-2

2456420

RCcysB-3

2485406

RCcysB-4

2415990

RCcysB-5

2498480

RCcysB-6

2442610

RCcysC-1

2537224

RCcysC-2

2502182

RCcysC-3

2514301

Mean

2479287

SD

38280

CV

1.5%

SD = Standard Deviation; CV = Coefficient of Variation.

Table 6

SPCC Area Ratio (A220/A258) of Reference Controls A, B and C

 

Reference Controls in

ACN

Peak area

at 220 nm (µAU)

Peak area

at 258 nm (µAU)

Area ratio (A220/A258)

RCcysA-1

2499196

136437

18.32

RCcysA-2

2489862

133983

18.58

RCcysA-3

2497917

137622

18.15

RCcysB-1

2460974

137687

17.87

RCcysB-2

2456420

139124

17.66

RCcysB-3

2485406

138536

17.94

RCcysB-4

2415990

132903

18.18

RCcysB-5

2498480

132808

18.81

RCcysB-6

2442610

132718

18.40

RCcysC-1

2537224

136421

18.60

RCcysC-2

2502182

136318

18.36

RCcysC-3

2514301

135985

18.49

 

Mean

18.28

Table 7

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

712190

0.140

71.7%

PCcys-2

778302

0.154

69.1%

PCcys-3

753856

0.149

70.1%

 

Mean

70.3%

SD

1.3%

 


SD = Standard Deviation.


Table 8


SPCC Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test ItemSamples

 

 

Sample code

Peak area at 220 nm

(µAU)

Concentration(mM)

SPCC

Depletion

Peak area

at 258 nm (µAU)

Area ratio(A220/A258)

209457/A-cys-1

2444865

0.513

0.0%

134492

18.18

209457/A-cys-2

2399294

0.503

1.0%

135057

17.77

209457/A-cys-3

2439721

0.512

0.0%

133001

18.34

 

Mean

0.3%

NA

18.10

SD

0.6%

NA

0.30

SD = Standard Deviation, NA = Not Applicable.

Table 9

SPCL Retention Times and Peak Areas (at 220 nm and 258 nm)

 

Sample code

Retention time of peak

at 220 nm (min)

Peak area at 220 nm

(µAU)

Peak area at 258 nm

(µAU)

STDlys7

NF

NF

NF

STDlys6

6.08

83099

6312

STDlys5

6.13

199459

11812

STDlys4

6.05

386135

21621

STDlys3

6.15

765184

42102

STDlys2

6.07

1379405

81624

STDlys1

6.00

2396719

153756

Dilution buffer

NF

NF

NF

RClysA-1

6.05

2398802

150899

RClysA-2

6.05

2433107

153366

RClysA-3

6.02

2498612

154811

CClys-209457/A

NF

NF

NF

RClysB-1

6.03

2469132

152648

RClysB-2

6.03

2490484

153564

RClysB-3

6.03

2413014

145604

RClysC-1

6.00

2257890

141816

RClysCMQ-1

6.05

2203475

138181

PClys-1

6.08

1203055

172428

209457/A-lys-1

6.03

2079535

135219

RClysC-2

5.98

2193380

139520

RClysCMQ-2

6.02

2183508

138208

PClys-2

5.98

1077706

167496

209457/A-lys-2

6.03

2107094

133717

RClysC-3

6.03

2205833

139824

RClysCMQ-3

6.03

2159567

138115

PClys-3

6.08

1032507

162773

209457/A-lys-3

6.02

2045330

126373

RClysB-4

6.00

2307430

142087

RClysB-5

5.98

2402779

150566

RClysB-6

5.97

2218517

139479

NF = No peak found.


Table 10

SPCL Peak Area at 220 nm and Concentration in Reference Controls A, C and CMQ

 

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RClysA-1

2398802

0.517

RClysA-2

2433107

0.524

RClysA-3

2498612

0.539

 

Mean

0.527

SD

0.011

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RClysC-1

2257890

0.486

RClysC-2

2193380

0.471

RClysC-3

2205833

0.474

 

Mean

0.477

SD

0.008

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

RClysCMQ-1

2203475

0.474

RClysCMQ-2

2183508

0.469

RClysCMQ-3

2159567

0.464

 

Mean

0.469

SD

0.005

 


SD = Standard Deviation.


Table 11


SPCL Peak Area of Reference Controls B and C

 

Reference Controls in ACN

Peak area at 220 nm (µAU)

RClysB-1

2469132

RClysB-2

2490484

RClysB-3

2413014

RClysB-4

2307430

RClysB-5

2402779

RClysB-6

2218517

RClysC-1

2257890

RClysC-2

2193380

RClysC-3

2205833

Mean

2328718

SD

116997

CV

5.0%

SD = Standard Deviation; CV = Coefficient of Variation.


Table 12

SPCL Area Ratio (A220/A258) of Reference Controls A, B and C

 

Reference Controls in

ACN

Peak area

at 220 nm (µAU)

Peak area

at 258 nm (µAU)

Area ratio (A220/A258)

RClysA-1

2398802

150899

15.90

RClysA-2

2433107

153366

15.86

RClysA-3

2498612

154811

16.14

RClysB-1

2469132

152648

16.18

RClysB-2

2490484

153564

16.22

RClysB-3

2413014

145604

16.57

RClysB-4

2307430

142087

16.24

RClysB-5

2402779

150566

15.96

RClysB-6

2218517

139479

15.91

RClysC-1

2257890

141816

15.92

RClysC-2

2193380

139520

15.72

RClysC-3

2205833

139824

15.78

 

Mean

16.03

Table 13

SPCL Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

 

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

SPCL Depletion

PClys-1

1203055

0.252

45.8%

PClys-2

1077706

0.224

51.4%

PClys-3

1032507

0.214

53.5%

 

Mean

50.2%

SD

4.0%

 


SD = Standard Deviation.


Table 14


SPCL Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test ItemSamples

 

 

Sample code

Peak area at 220 nm

(µAU)

Concentration(mM)

SPCL

Depletion

Peak area

at 258 nm (µAU)

Area ratio(A220/A258)

209457/A-lys-1

2079535

0.446

4.7%

135219

15.38

209457/A-lys-2

2107094

0.452

3.4%

133717

15.76

209457/A-lys-3

2045330

0.439

6.3%

126373

16.18

 

Mean

4.8%

NA

15.77

SD

1.4%

NA

0.40

SD = Standard Deviation, NA = Not Applicable.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, this DPRA test is valid. Riboxyl TM 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 objective of this study was to determine the reactivity of RiboxylTM 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 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.

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

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability

criteria

Results for

SPCC

Acceptability

criteria

Results for

SPCL

Correlation coefficient(r2)

standard calibrationcurve

>0.99

0.996

>0.99

0.992

Mean peptide concentration

RC-A samples (mM)

0.50 ± 0.05

0.524 ± 0.001

0.50 ± 0.05

0.527 ± 0.011

Mean peptide concentration

RC-C samples (mM)

0.50 ± 0.05

0.529 ± 0.004

0.50 ± 0.05

0.477 ± 0.008

Mean peptide concentration

RC-CMQsamples (mM)

0.50 ± 0.05

0.508 ± 0.008

0.50 ± 0.05

0.469 ± 0.005

CV (%) for RC samples

B and C

<15.0

1.5

<15.0

5.0

Mean peptidedepletion

cinnamic aldehyde(%)

60.8-100

70.3

40.2-69.0

50.2

SD of peptidedepletion

cinnamic aldehyde(%)

<14.9

1.3

<11.6

4.0

SD of peptide depletion for the test item (%)

<14.9

0.6

<11.6

1.4

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

The validation parameters, i.e. calibration curve, mean concentration of Reference Control(RC) samples A, C and CMQ, the CV for RC samples B and C, the mean percent peptidedepletion 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.

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

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.Inthe cysteine reactivity assay the test item showed 0.3% SPCC depletion while in the lysine reactivity assay the test item showed 4.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.6% 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.


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

RiboxylTM

0.3%

±0.6%

4.8%

±1.4%

2.6%

Negative: No or minimal reactivity

SD = Standard Deviation.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. RiboxylTMwas 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.