Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 - 19 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

yes

Remarks:

no information on vehicle control and reference substances provided

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:

Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

EC Number:

942-424-7

Molecular formula:

not applicable (reaction mass)

IUPAC Name:

Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

In chemico test system

Details on the study design:

TEST SYSTEM
- Supplier: AnaSpec
- Cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 90 - 95%
Expiry date: 5 years

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.

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Supplier: SAFC
- 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.

POSITIVE CONTROL PREPARATION
The positive control was prepared at a concentration of 100 mM.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile.

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); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h

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
- Injection volume: 2 µL
- Column temperature: 30 °C

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of cysteine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

-0.996

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

11.5

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % overall mean depletion

Value:

5.23

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile 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. Mean peptide depletion of cysteine-containing peptide.

	Peak area (µV.sec)	Peptide concentration [µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	949469	383	-1.01	-0.996 ± 0.06
	948717	383	-0.927
	949895	383	-1.05
Positive control	234824	96.3	75.0	74.0 ± 3.77
	243906	100	74.1
	253197	104	73.1

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

**: Calculated against a mean reference control peak area of 940000 μV.sec (n=6)

Table 5. Mean peptide depletion of lysine-containing peptide.

	Peak area (µV.sec)	Peptide concentration [µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	703216	345	10.8	11.5 ± 0.63
	697085	342	11.6
	694715	341	11.9
Positive control	341222	167	56.7	55.8 ± 7.06
	376020	184	52.3
	328441	161	58.4

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

**: Calculated against a mean reference control peak area of 788680 μV.sec (n=6)

Applicant's summary and conclusion

Interpretation of results:

other: DPRA prediction: negative (no or minimal reactivity)

Remarks:

according to OECD TG 442C

Conclusions:

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

Executive summary:

Solutions of test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.