Carnosine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 May 2017 - 27 May 2017

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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Carnosine

EC Number:

206-169-9

EC Name:

Carnosine

Cas Number:

305-84-0

Molecular formula:

C9H14N4O3

IUPAC Name:

(2S)-2-(3-aminopropanoylamino)-3-(1H-imidazol-4-yl) propanoic acid

In chemico test system

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 94%
Expiry date: 5 years

VEHICLE CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Vehicle (positive control): acetonitrile
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
- Vehicle (test item): water
The solubility of the test substance in a series of solvents was assessed at a concentration of 100 mM. A stock solution of the test substance was prepared at 100 mM in water.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: > 95%
- Expiry date: Feb 2019

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C
- Sample temperature: 25 °C

Results and discussion

In vitro / in chemico

Results

Results 1

Parameter:

other: % depletion of cystein-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

0.075

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

1

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 3

Key result

Parameter:

other: % overall mean depletion

Value:

0.536

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution peaks were observed in either the Cysteine or Lysine assay. The solubility of the test substance in water at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Any other information on results incl. tables

Table 5: Depletion of cysteine-containing peptide

Sample	Peak area (µV.sec)	Peptide concentration^*(µg/mL)	Peptide Depletion^**(%)	Mean Depletion (%)	SD (%)
Positive control	256166	112.78	70.1	70.0^***	0.30
	256241	112.81	70.1
	260636	114.75	69.6
Test substance^***	858192	378.14	-0.481	0.0750	0.61
	847846	373.58	0.730
	854295	376.43	-0.0250

SD    Standard Deviation

^*             Samples prepared at a concentration of 376 µg/mL (0.5 mM)

^**           Calculated against a mean Reference Control area of 857880 µV.sec(n=6)

^***          Individual and mean concentration values are outside of the range set in the historic data (Annex 1). This is considered not to have impacted on the data reported as the mean depletion value is within acceptance criteria limits.

^****        Calculated against a mean Control C peak area of 854080 µV.sec(n=3)

Table 6: Depletion of lysine-containing peptide

Sample	Peak area (µV.sec)	Peptide concentration^*(µg/mL)	Peptide Depletion^**(%)	Mean Depletion (%)	SD (%)
Positive control	305750	158.56	60.9	59.8	0.99
	316448	163.91	59.6
	320860	166.12	59.0
Test substance^***	766486	389.04	0.113	1.00	1.07
	762062	386.82	0.689
	750596	381.09	2.18

SD   Standard Deviation

^*             Samples prepared at a concentration of 388 µg/mL (0.5 mM)

^**      Calculated against a mean Reference Control area of 782340 µV.sec(n=6)

^***     Calculated against a mean Control C peak area of 767350 µV.sec(n=3)

 

Applicant's summary and conclusion

Interpretation of results:

other: DPRA prediction: negative (no or minimal peptide reactivity) according to OECD 442C

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.

Executive summary:

Protein reactivity of the test substance was determined in a Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (2017). The overall mean percentage of cystein and lysine depletion was 0.536% and therefore the test substance showed no or minimal peptide reactivity (mean % depletion ≤ 6.38%).