2-Cyclohexen-1-one, 2-methyl-5-propyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation

Remarks:

other: under validation "in chemico" test method

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-01-08 to 2014-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), assessed on 13 November 2013

Deviations:

no

Principles of method if other than guideline:

The study was performed according to the methods described in the following publications:
- 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 non-animal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.
- Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro
methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504.

In addition the study is performed according to the methods described in the following publications:
- 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. Quantification of Chemical Peptide Reactivity for Screening Contact Allergens: A
Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-12-18

Type of study:

other: Direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-5-propylcyclohex-2-en-1-one

EC Number:

807-489-7

Cas Number:

1447712-18-6

Molecular formula:

C10H16O1

IUPAC Name:

2-methyl-5-propylcyclohex-2-en-1-one

Test material form:

other: liquid

Details on test material:

- Physical state: liquid, clear pale yellow

In vivo test system

Test animals

Details on test animals and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Vehicle:

other: acetonitrile

Induction 2

Concentration / amount:

- 0.5 mM cysteine-containing peptide with 5 mM test substance (ratio of 1:10)
- 0.5 mM lysine-containing peptide with 25 mM test substance (ratio of 1:50)

Challenge

Challenge 1

Vehicle:

other: acetonitrile

Challenge 2

Concentration / amount:

- 0.5 mM cysteine-containing peptide with 5 mM test substance (ratio of 1:10)
- 0.5 mM lysine-containing peptide with 25 mM test substance (ratio of 1:50)

No. of animals per dose:

test item: triplicate (for each peptide)
vehicle control: triplicates
co-elution control: triplicates

Details on study design:

TEST SUBSTANCE PREPARATION
- test substance was prepared as a 100 mM solution in acetonitrile within 4 hours of preparation of test-substance samples. After short stirring the test substance was soluble in the vehicle.
- no analysis of the test substance in the vehicle was performed because the test-substance preparation was incubated with the peptide (DPRA) shortly after preparation.

EXPERIMENTAL PROCEDURE
1) Preparation of peptide stock solutions:
- peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (cysteine-containing peptide) or pH 10.2 ammonium acetate buffer (lysine-containing peptide).
- peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

2) Preparation of calibration samples:
- calibration samples were prepared from the peptide stock solutions in 20% acetonitrile:buffer (= dilution buffer) using serial dilution: 0.534, 0.267, 0.1335, 0.0667, 0.0334, and 0.0167 mM peptide plus 0.000 dilution buffer
- analysis of the calibration samples was started before analysis of the test-substance samples.

3) Preparation of the test-substance samples:
- three samples of the test substance were incubated with each of two peptides, cysteine- (C-) containing peptide and lysine- (K-) containing peptide (peptides contained phenylalanine to aid in detection and either cysteine or lysine as the reactive center).
- 0.5 mM cysteine-containing peptide was incubated with 5 mM test substance (ratio of 1:10) and 0.5 mM lysine-containing peptide with 25 mM test substance (ratio of 1:50).
- samples were incubated at room temperature in the dark for 24 ± 2 hours.
- prior to HPLC analysis (HPLC with gradient elution and UV-detection at 220 nm (Liquid chromatograph Waters 2695 Seperation Module with DAD)), samples were visually investigated for any precipitate that may have formed during the exposure period. As the samples of the K-peptide were visually turbid they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles.
- about 24 hours after sample preparation HLPC analysis started. The analysis time did not exceed 30 hours for the samples of the C-peptide but for the samples of the K-peptide. As stability of the K-peptide was demonstrated over the analysis time, this deviation was not considered to have an influence on the result of the study.

4) Preparation of the vehicle controls:
- several acetonitrile controls were prepared in triplicates in the same way as the test substance samples but with acetonitrile instead of the test substance:
Set A (performance control): analyzed together with the calibration samples without incubation
Set B (two sets; stability control of the peptide over the analysis time): sets which were incubated with the samples were placed at the very start and ending of the sample list
Set C: analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in acetonitrile.

5) Preparation of the co-elution control:
- co-elution control was prepared in the same way as the test-substance samples but without the peptide and with peptide buffer.
- as the sample in pH 10.2 ammonium acetate buffer were visually turbid precipitates they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles.

6) Measurement of peptide concentrations:
- 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, and 25/10
Wavelength: 220 nm and 258 nm
Injection volume: 2 μL
HPLC: Waters 2695 Seperation Module
Detection: Diode Array Detector
Software: Dionex Chromeleon

DATA EVALUATION
- some test substances or reaction products may co-eluate with the peptides. In these cases where proper integration and calculation of peptide depletion was not possible, the result for the respective peptide is reported as interference.
- when samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 / 258 may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples.
- due to small peak areas calculation of the area ratio may not be possible for all samples.
- calculation of the peptide concentrations: for each peptide a calibration curve was generated from the measured peak areas of the calibration samples of known peptide concentration.
The peptide concentration of the samples was calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Peptide concentration [mM] = (peak area [mAU x s] - b)/m

- calculation of the peptide depletion: the peptide depletion of a sample was calculated as follows:
Peptide depletion of sample =[1 - (peptide concentration of sample (mM)/mean peptide concentration of negative control (mM))] x 100 [%]
The mean peptide depletion for each of the two peptides was calculated as the mean value of the three samples conducted for each peptide and test substance (C-containing and K-containing peptide depletion; example calculation for C-containing peptide):
C-containing peptide depletion of a test substance [%] = mean [C-containing peptide depletion of samples 1 - 3][%]
If several valid test runs were performed the mean value of these runs will be determined.
When a negative value for C- or K-peptide depletion was obtained the value was considered zero for calculation of the mean peptide depletion.
The mean peptide depletion of a test substance was calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion:
Mean peptide depletion [%] = (C-containing peptide depletion [%] + K-containing peptide depletion [%])/2

ACCEPTANCE CRITERIA
- if any of the acceptance criteria was not met, repetition of the test was considered.
- study is considered acceptable if the positive control causes depletion of both peptides comparable to historical data.
- negative control (vehicle control) samples analyzed should be 0.50 mM ± 0.05 mM.
- Coefficient of variation (CV) of the nine vehicle controls B and C should be < 15%.
- since the mean peptide depletion for each peptide was determined from the mean of three single samples, the variability between these samples should be acceptably low.

EVALUATION
Chemical reactivity was determined by mean peptide depletion [%] and was rated as high, moderate, low, or minimal (according to the classification tree model described by Gerberick et al.):
> 42.47%: high reactivity
> 22.62% < 42.47%: moderate reactivity
> 6.38% < 22.62%: low reactivity
< 6.38%: minimal reactivity

Positive control substance(s):

yes

Remarks:

Ethylene glycol dimethacrylate (prepared as a 50 mM solution in acetonitrile)

Results and discussion

Positive control results:

Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

RESULTS

CYSTEINE-CONTAINING PEPTIDE

Cysteine-peptide vehicle controls in acetonitrile:

- mean peptide concentration of the three samples of set A was calculated to be 0.508 mM with a SD of 0.020, demonstrating good performance.

- mean peptide concentration of the three samples of set B, analyzed at the beginning of the sample list was calculated to be 0.515 mM with a SD of 0.014. The other samples of set B (two samples only due to a technical error), analyzed at the end of the sample list had a mean peptide concentration of 0.483 mM with a SD of 0.007. Thus the peptide was considered stable over the time of analysis.

- CV of the vehicle control samples of sets B and C was calculated to be 3.7%.

Reaction with cysteine-peptide

Table 1: Peak area, peptide concentration and peptide depletion of negative control, positive control and the test substance.

Reaction with cysteine-peptide	Peak area [mAU*s] at 220 nm			Peptide concentration [mM]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD
Negative control: acetonitrile	485.5	460.6	455.9	0.512	0.486	0.481	0.493	0.017
Test item	327.5	297.7	284.7	0.346	0.315	0.302	0.321	0.023
Positive control: Ethylene glycol dimethacrylate in acetonitrile	240.2	199.9	51.5	0.255	0.213	0.058	0.175	0.104

Reaction with cysteine-peptide	Peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Mean	SD
Negative control: acetonitrile	-3.85	1.43	2.43	0.00	3.38
Test item	29.69	36.02	38.78	34.83	4.66
Positive control: Ethylene glycol dimethacrylate in acetonitrile	48.24	56.78	88.30	64.44	21.10

Table 2: Area ratio 220/258 of negative control, positive control and the test substance.

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	13.2	12.5	12.4	36.9	36.7	36.8
Test item	8.8	8.1	7.6	37.4	36.7	37.6
Positive control: Ethylene glycol dimethacrylate in acetonitrile	n.a.	n.a.	n.a.	-	-	-

LYSINE-CONTAINING PEPTIDE

Lysine-peptide vehicle controls in acetonitrile:

- mean peptide concentration of the three samples of set A was calculated to be 0.511 mM with a SD of 0.001, demonstrating good performance.

- mean peptide concentration of the three samples of set B, analyzed at the beginning of the sample list was calculated to be 0.496 mM with a SD of 0.005. The other three samples of set B, analyzed at the end of the sample list had a mean peptide concentration of 0.495 mM with a SD of 0.006. Thus the peptide was considered stable over the time of analysis.

- CV of the vehicle control samples of sets B and C was calculated to be 1.0%.

Reaction with lysine-peptide

Table 3: Peak area, peptide concentration and peptide depletion of negative control, positive control and the test substance.

Reaction with lysine-peptide	Peak area [mAU*s] at 220 nm			Peptide concentration [mM]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD
Negative control: acetonitrile	415.3	422.4	425.8	0.490	0.498	0.502	0.497	0.006
Test item	419.3	412.6	411.1	0.494	0.486	0.485	0.488	0.005
Positive control: Ethylene glycol dimethacrylate in acetonitrile	329.7	331.5	324.3	0.388	0.391	0.382	0.387	0.004

Reaction with lysine-peptide	Peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Mean	SD
Negative control: acetonitrile	1.40	-0.30	-1.10	0.00	1.28
Test item	0.45	2.05	2.39	1.63	1.04
Positive control: Ethylene glycol dimethacrylate in acetonitrile	21.77	21.33	23.05	22.05	0.89

Table 4: Area ratio 220/258 of negative control, positive control and the test substance.

Reaction with lysine-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	12.4	12.5	12.7	33.4	33.9	33.4
Test item	11.7	12.1	11.5	35.8	34.1	35.7
Positive control: Ethylene glycol dimethacrylate in acetonitrile	9.5	9.4	9.6	34.9	35.4	33.7

ADDITIONAL OBSERVATIONS

- test substance was soluble in acetonitrile.

- samples of the test substance with the peptides were solutions (C-peptide) or emulsions (K-peptide).

- after 24 hours no precipitates were noticed in all samples, but the samples of the K-peptide were emulsions, still.

- no co-elution of the test substance and peptides occurred.

MEAN PEPTIDE DEPLETION

Table 5: Mean peptide depletions of cysteine, lysine and both peptides

	Cysteine-Peptide		Lysine-Peptide		Mean of both depletions [%]
	Mean depletion [%]	SD	Mean depletion [%]	SD
Test item	34.83	4.66	1.63	1.04	18.23
Positive control: Ethylene glycol dimethacrylate in acetonitrile	64.44	21.10	22.05	0.89	43.25

HISTORICAL CONTROL DATA

Table 6: Historical range of negative control (acetonitrile)

Historical Period	mean peptide concentration [mM]	SD of peptide concentration
Jan 2012 - Dec 2013 (no of tests performed: 38)
Cysteine-peptide	0.485	0.027
Lysine-peptide	0.503	0.016

Table 7: Historical range of positive control (ethylene glycol dimethacrylate 98% (50 mM in acetonitrile)

Historical Period	mean peptide concentration [mM]	SD of peptide concentration	mean peptide-depletion [%]	SD of peptide-depletion [%]
Feb 2012 - Dec 2013 (no of tests performed: 35)
Cysteine-peptide	0.196	0.050	60	10
Lysine-peptide	0.441	0.028	13	5

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The mean peptide depletion of the test item obtained during testing was 18.23 %. It was concluded that the test item showed a low chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

The reactivity of the test item towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). Three samples of the test substance was incubated with each synthetic peptide 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. Additionally triplicates of the concurrent vehicle control 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 samples of the test substance with the peptides were solutions (C-peptide) or emulsions (K-peptide). After 24 hours no precipitates were noticed in all samples, but the samples of the K-peptide were emulsions, still. Thus the samples and the co-elution control of the K-peptide were centrifuged prior to HPLC analysis.

The mean C-peptide depletion, caused by the test substance was determined to be 34.83%.

The mean K-peptide depletion, caused by the test substance was determined to be 1.63%.

Thus, the mean peptide depletion was calculated to be 18.23%.

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 item shows a low chemical reactivity in the DPRA under the test conditions chosen.