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Diss Factsheets

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:
The study was conducted between 08 January 2019 and 11 January 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discriminating between skin sensitizing and non-sensitizing chemicals and thus assesses their sensitization potential.

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one
EC Number:
950-718-1
Molecular formula:
C14H22O
IUPAC Name:
Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one
Test material form:
liquid
Specific details on test material used for the study:
Identification PG-RAW-90-032
Appearance: Clear liquid
Storage conditions: Refrigerated temperature (2°C to 8°C)

In chemico test system

Details on the study design:
Peptide and Positive Control
Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1857724
Stated purity: 96.2% (by HPLC)
Molecular Weight: 751 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch number: 1658141
Stated purity: 96.8% (by HPLC).
Molecular Weight: 776 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Cinnamic Aldehyde (Positive control)
Batch number: MKCB9907
Stated purity: 99.1%
Molecular Weight: 132 g/mol
Supplier: SAFC
Storage conditions: Room temperature (15°C to 25°C)
Expiry/retest date: November 2021

Apparatus
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Analytical Procedure
Reagents
Acetonitrile (ACN): HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

Assessment of Test Item Solubility
The solubility of PG-RAW-90-032 was assessed in acetonitrile at a nominal concentration of 100 mM.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of PG-RAW-90-032 was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and PG-RAW-90-032 stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM PG-RAW-90-032. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and PG-RAW-90-032 stock solution were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM PG-RAW-90-032. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the PG-RAW-90-032 and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of PG-RAW-90-032 and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes) MP A (%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes

Results and discussion

In vitro / in chemico

Results
Run / experiment:
mean
Parameter:
other: % Overall mean peptide depletion (reactivity)
Value:
0.622
Positive controls validity:
valid

Any other information on results incl. tables

Solubility Assessment

Solubility of PG-RAW-90-032 was achieved at a nominal concentration of 100 mM in acetonitrile.

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

Peptide

Standard Linearity

Positive control depletion (%)

Reference controls

Test item

Acceptance criteria

Cysteine

r2>0.99

60.8-100
(SD <14.9%)

0.45-0.55 mM (CV <15%)

SD <14.9%

Lysine

r2>0.99

40.2-69.0
(SD <11.6%)

0.45-0.55 mM (CV <15%)

SD <11.6%

Achieved results

Cysteine

r2>0.999

71.6
(SD, 0.16%, n=3)

B: 0.500 mM (CV 0.48%, n=6)

SD 0.12% (n=3)

Lysine

r2>0.999

57.1
(SD, 0.68%, n=3)

B: 0.500 mM (CV 0.66%, n=6)

SD 0.67% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of PG-RAW-90-032 was:

Peptide

Reference Control

Mean peak area of peptide with test item(µV.sec)

Mean peptide depletion by
PG-RAW-90-032 (%)

Cysteine

Control B: 924280 (n=6)

912780 (n=3)

1.24

Lysine

Control B: 797070 (n=6)

807570 (n=3)

-1.32

Applying the following reactivity prediction depletion model (below), reactivity of PG-RAW-90-032 is classed as “no or minimal” and the DPRA prediction is therefore negative and is therefore predicted not to be a potential skin sensitizer. 

Mean of cysteine and lysine% depletion

Reactivity Class

DPRA Prediction

0%≤ mean% depletion ≤6.38%

No or minimal reactivity

Negative

6.38%< mean% depletion ≤22.62%

Low reactivity

Positive

22.62%< mean% depletion ≤42.47%

Moderate reactivity

42.47%< mean% depletion ≤100%

High reactivity

There were no co-elution peaks in either the Cysteine or Lysine assay.   

Overall Achieved Depletion Values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

PG-RAW-90-032

1.24

0.001

0.622

Minimal

Negative

1          For calculation of overall mean depletion a negative value counts as zero

Individual Achieved Depletion Values

Cysteine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD
 (%)

Positive control

260938

106

71.8

71.6

0.16

263778

107

71.5

263076

107

71.5

PG-RAW-90-032

911464

370

1.39

1.24

0.12

913600

371

1.16

913262

371

1.19

SD      Standard Deviation

1        Samples prepared at a nominal concentration of 376 µg/mL (0.5 mM)

2        Calculated against a mean Reference Control (B) area of 924280 µV.sec (n=6)

Lysine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD
 (%)

Positive control

336270

164

57.8

57.1

0.68

341481

167

57.2

347028

169

56.5

PG-RAW-90-032

808183

394

-1.39

-1.32

0.67

812568

396

-1.94

801959

391

-0.613

SD      Standard Deviation

1         Samples prepared at a nominal concentration of 388 µg/mL (0.5 mM)

2         Calculated against a mean Reference Control (B) area of 797070 µV.sec (n=6)

Applicant's summary and conclusion

Interpretation of results:
other: Predicted to be negative
Remarks:
Criteria used: EU CLP
Conclusions:
Solutions of PG-RAW-90-032 were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 0.622% in the presence of the test item, PG-RAW-90-032 is only minimally reactive and is predicted by DPRA as negative and therefore unlikely to be a potential skin sensitizer based on this assay.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of PG-RAW-90-032. 

Solutions of PG-RAW-90-032 were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. There was minimal reactivity of both peptides in the presence of PG-RAW-90-032. With an overall depletion of peptide of 0.622% the reactivity of PG-RAW-90-032 is henceforth classified as “no or minimal” therefore the DPRA prediction is negative. PG-RAW-90-032 is likely not to be a potential skin sensitizer based on this assay.