Methyl benzoylformate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Although the study predates the adoption of the OECD 442 C guideline (4 Feb 2015) it was conducted to the methods described in the following literature sources, and used as the basis for the OECD 442 C guideline:
• Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.
• Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
• Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.
• Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.
The study report contains no deviations from the OECD 442 C test method.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl benzoylformate

EC Number:

239-263-3

EC Name:

Methyl benzoylformate

Cas Number:

15206-55-0

Molecular formula:

C9H8O3

IUPAC Name:

methyl oxo(phenyl)acetate

In chemico test system

Details on the study design:

The test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

Test system:
Synthetic peptides:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

HPLC:
- Liquid chromatograph: Agilent HP 1100 with DAD
- Software: Dionex Chromeleon
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column "Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- Pipettes / positive displacement pipette: For pipetting liquids of different viscosity up to 5 mL and graduated pipette with pipettor or graduated cylinder for higher volumes.
- pH meter: Readability +/- 0.1 pH units.
For adjusting pH-values of buffers.

- HPLC mobile phase A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
- HPLC mobile phase B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade

- Reagents for preparing the buffers:
Sodium phosphate, monobasic monohydrate, CAS-no. 10049-21-5 (e.g. Sigma-Aldrich S9638)
Sodium phosphate, dibasic heptahydrate, CAS-no. 7782-85-6 (e.g. Sigma-Aldrich S9390)
Ammonium acetate, CAS-no. 631-61-8 (e.g. Sigma-Aldrich 32301)
Ammonium hydroxide, 28% – 30%, CAS-no. 1336-21-6 (e.g. Sigma-Aldrich 320145)

Controls:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) (prepared as a 50 mM solution in acetonitrile)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test-substance preparation:
The test substance solutions were prepared within 4 hours of performing the assay (preparation of samples).
- Test-substance preparation: The test substance was prepared as a 100 mM solution in acetonitrile. After short stirring the test substance was soluble
in the vehicle.
- Vehicle: acetonitrile
- Reason for the vehicle: The test substance was soluble in acetonitrile.

Measurement of peptide concentrations:
The analyses of the samples were performed via HPLC under the following conditions:
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column "Security Guard“ C18, 4 mm x 2 mm
- Eluent:
A: 0.1% (v/v) trifluoracetic acid in de-ionized water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
time [min] %B
0 10
10 25
11 90
13 90
13.5 10
25 10
- Wavelength: 220 nm and 258 nm
- Injection volume: 2 µL

Results and discussion

Positive control results:

See "Any other information on results incl. tables".

In vitro / in chemico

Results

Results 1

Parameter:

other: Mean C-peptide depletion [%]

Value:

3.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Parameter:

other: Mean K-peptide depletion [%]

Value:

19.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test was determined to be valid. See below for full result tables and historical controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: See below for full result tables and historical controls.

Any other information on results incl. tables

RESULTS WITH CYSTEINE-PEPTIDE

Reaction with cysteine-peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]			mean	SD
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	744.3	753.2	741.0	0.491	0.497	0.489	0.492	0.004
Test material	717.0	726.5	723.7	0.473	0.479	0.477	0.476	0.003
Positive control - Ethylene glycol dimethacrylate	356.0	320.8	2655	0.235	0.212	0.176	0.208	0.03

Reaction with cysteine-peptide	peptide depletion [%]			mean	SD
	sample 1	sample 2	sample 3
Negative control - Acetonitrile	0.24	-0.94	0.7	0.0	0.84
Test material	3.9	2.64	3.0	3.18	0.65
Positive control - Ethylene glycol dimethacrylate	52.18	56.88	64.28	57.78	6.1

Reaction with cysteine-peptide	peak area [mAU*s] at 258 nm			area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	20.9	21.4	20.8	35.5	35.2	35.7
Test material	20.9	20.9	19.5	34.4	34.8	37.1
Positive control - Ethylene glycol dimethacrylate	9.5	8.6	6.7	37.5	37.5	39.4

RESULTS WITH LYSINE-PEPTIDE

Reaction with cysteine-peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]			mean	SD
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	722.0	728.5	721.8	0.503	0.507	0.502	0.504	0.003
Test material	583.8	588.9	576.1	0.407	0.41	0.403	0.407	0.004
Positive control - Ethylene glycol dimethacrylate	624.1	620.0	610.5	0.435	432	0.425	0.431	0.005

Reaction with cysteine-peptide	peptide depletion [%]			mean	SD
	sample 1	sample 2	sample 3
Negative control - Acetonitrile	0.29	-0.6	0.32	0	0.52
Test material	19.31	18.62	20.09	19.34	0.74
Positive control - Ethylene glycol dimethacrylate	13.76	14.33	15.64	14.58	0.96

Reaction with cysteine-peptide	peak area [mAU*s] at 258 nm			area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	21.0	21.3	21.1	34.3	34.2	34.2
Test material	17.0	17.0	16.5	34.4	34.6	35.0
Positive control - Ethylene glycol dimethacrylate	18.4	17.7	17.7	33.9	35	34.5

HISTORICAL CONTROL DATA

Historical Range of NC

Acetonitrile

Historical Period	mean peak area [mAU*s]	mean peptide concentration [mM]	SD of peptide concentration
Jan - Feb 2013 (no of tests performed: 29)
Cysteine-peptide	779.0	0.487	0.0201
Lysine-peptide	701.0	0.503	0.031

Historical Range of PC

Ethylene Glycol dimethacrylate 98% (50 mM in ACN)

Historical Period	mean peak area [mAU*s]	mean peptide concentration [mM]	SD of peptide concentration	mean peptide depletion [%]
Feb 2012 - Feb 2013 (no of tests performed: 25)
Cysteine-peptide	312	0.194	0.057	60
Lysine-peptide	625	0.448	0.026	11

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean depletion % to the test material were determined to be C-peptide 3.18% and K-peptide 19.34%. The test material was determined to be of low reactivity under the conditions of the test.

Executive summary:

The reactivity of the test material towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at

room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

The test substance was solved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

The mean C-peptide depletion, caused by the test substance was determined to be 3.18%.

The mean K-peptide depletion, caused by the test substance was determined to be 19.34%.

Thus, the mean peptide depletion was calculated to be 11.26%.

No co-elution of test substance and peptides was noticed.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) and cited in chapter 3.10 it was concluded that the test material shows a low chemical reactivity in the DPRA under the test conditions chosen.