2-octyldodecyl 5-oxo-L-prolinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 03, 2017 to July 14, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Test material

Test material information

Constituent 1

Reference substance name:

2-octyldodecyl 5-oxo-L-prolinate

EC Number:

253-604-3

EC Name:

2-octyldodecyl 5-oxo-L-prolinate

Cas Number:

37673-37-3

Molecular formula:

C25H47NO3

IUPAC Name:

2-octyldodecyl 5-oxo-L-prolinate

Test material form:

liquid

In chemico test system

Details on the study design:

See below 'Any other information for materials and methods incl. tables' for details on study design.

Reference Substance
Reference Substance Name: Cinnamic aldehyde, kosher Test Facility Test Item Number: RS473/A
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKBP1014V
Purity: 98.4%
Test item storage: In the refrigerator (2-8°C)
Stable under storage conditions until: 31 May 2018
Purity/composition correction factor: Yes

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 3

Parameter:

other: % Mean Cysteine Peptide Depletion

Value:

7.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Run / experiment:

other: 3

Parameter:

other: % Mean Lysine Peptide Depletion

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 3 each Cys and Lys

Parameter:

other: Mean % Peptide Depletion (Cys + Lys)

Value:

5.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Since precipitation was observed in both peptide solutions (Cys and Lys), amount of test substance that remained in the solution to react with the peptides is unknown. Consequently, this negative result is uncertain.

Other effects / acceptance of results:

All acceptance/validation criteria were met.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and C(isopropanol), the Coefficient of Variation 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 substance, were all within the acceptability criteria for the DPRA.

The means of Reference and Positive Control samples in either of Cysteine or Lysine Reactivity Assay were within the acceptance criteria, this confirms the suitability of the HPLC system and indicates that the solvent used to dissolve the test substance did not impact the % Peptide Depletion.

The area ratio (A220/A258) of the Reference Control samples in either of Cysteine or Lysine Reactivity Assay were within range (12.77-19.59) giving indication that no co-elution occurred.

Any other information on results incl. tables

Results

 

Solvent Selection

Isopropanol was found to be an appropriate solvent to dissolve the test substance and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

Acceptance criteria

Acceptance criteria for all controls and the test substance were met in both Cysteine or Lysine Reactivity Assay 

Acceptability of the Direct Peptide Reactivity Assay (DPRA)
	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability	Results for	Acceptability	Results for
	criteria	SPCC	criteria	SPCL
Correlation coefficient (r2)	>0.99	0.991	>0.99	0.994
standard calibration curve
Mean peptide concentration	0.50 ± 0.05	0.517 ± 0.008	0.50 ± 0.05	0.521 ± 0.013
RC-A samples (mM)
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.511 ± 0.008	0.50 ± 0.05	0.528 ± 0.009
Mean peptide concentration	0.50 ± 0.05	0.468 ± 0.006	0.50 ± 0.05	0.522 ± 0.017
RC-Cwatersamples (mM)
CV (%) for RC samples	<15.0	1.4	<15.0	2.5
B and C
Mean peptide depletion	60.8-100	71.2	40.2-69.0	50.5
cinnamic aldehyde (%)
SD of peptide depletion	<14.9	0.2	<11.6	1.8
cinnamic aldehyde (%)
SD of peptide depletion for the test substance (%)	<14.9	1.2	<11.6	2.5
RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Results Summary

The test substance produced 5.20% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. However, since precipitation was observed upon addition of the test substance to the peptide solutions, Amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test substance
SPCC		SPCL		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
depletion		depletion
Mean	± SD	Mean	± SD	5.20%	Cysteine 1:10 / Lysine 1:50 prediction model
7.10%	±1.2%	3.30%	±2.5%		Negative: No or minimal reactivity
SD = Standard Deviation

 

Applicant's summary and conclusion

Conclusions:

the test substance was determined to be non-sensitising. However, since precipitation was observed upon addition of the test substance to the peptide solutions, the amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care.

Executive summary:

A study was conducted to determine the skin sensitisating potential of the test substance according to OECD Guideline 442C, in chemico Direct Peptide Reactivity Assay (DPRA), in compliance with GLP. Test samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) at 25 ± 2.5˚C for 24 - 30 h. After incubation, the relative peptide concentration was determined by HPLC using gradient elution and photodiode array (PDA) detector to measure peptide concentration. Test substances were compared to reference controls in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes for skin sensitisation. Acceptance criteria for all controls and the test substance were met in either of Cysteine or Lysine Reactivity Assay. The test substance produced 5.2% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitising with no or minimal reactivity. Under study conditions, the test substance was determined to be non-sensitising. However, since precipitation was observed upon addition of the test substance to the peptide solutions, the amount of test substance that remained in the solution to react with the peptides was unknown. Consequently, the negative result was determined to be uncertain and should be interpreted with due care (Reinen, 2017).