ethyl 2-[[(1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexanecarbonyl]amino]acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

EC Number:

695-735-2

Cas Number:

68489-14-5

Molecular formula:

C15H27NO3

IUPAC Name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: white crystalline powder, WS-5, batch no.: 80100039
- Expiration date of the batch: 16 January 2019
- Purity test date: 99.57%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry ambient temperature

The test article, WS-5 produced a visually clear solution at a concentration of 100 mM acetonitrile, which was the first of the listed vehicles specified in the protocol.
Formulations were prepared shortly before testing.

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Test Article Incubation:
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24±2 hours, in the dark. At the end of the incubation period the samples were visually inspected for precipitate formation. Samples were centrifuged at 400 g for 5
minutes.

Analytical Method:
The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography
(HPLC) with gradient elution and UV detection at 220 nm.

Reference and Co-elution Controls:
Reference controls were prepared for each peptide.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the
stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides.

Calibration Curves for Peptides:
Calibration curves were prepared for each peptide using concentrations of 0.0167, 0.0334, 0.0667, 0.1335, 0.267 and 0.534 mM (Standards 1 to 6)

Results and discussion

Positive control results:

Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 98.5%, expiry 29 February 2020) was used as the positive control. The positive control was dissolved in acetonitrile at a concentration of 100 mM. For results, please see table 2 and 3.

In vitro / in chemico

Results

Key result

Run / experiment:

other: 3

Parameter:

other: The mean of cysteine and lysine percentage depletion

Value:

3.73

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

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

Remarks:

Negative prediction in the Direct Peptide Reactivity Assay

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean percent cysteine and percent lysine depletion value was calculated. Negative depletion was considered as “0” when calculating the mean. By using the
cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.

The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.

ACCEPTANCE OF RESULTS:
The following criteria should be met for a run to be considered valid:

- The standard calibration curve should have a r2 >0.99.
The r value for the standard calibration curve was 1.000 and as 0.9963 for lysine and cyteine depletion, respectively.
- The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference
controls B and C should be <15.0%.
For lysine and cysteine depletion, the mean Peptide Concentration (mM) for the Reference Controls A and C were as follows:
Reference Controls A - 0.50
- The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges:

Peptide Mean PPD Values (%) Lower Bound Mean PPD Values (%) Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6

- The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
The SD for Mean PPD for lysine and cysteine depletion were 0.81 and 2.72 , respectively.

Any other information on results incl. tables

Table 1. Cysteine 1:10/lysine 1:50 Prediction Model used in this testing to discriminate between skin sensitisers and non-sensitisers in the framework of an IATA

Mean of Cysteine and Lysine % Depletion	Reactivity Class	DPRA Prediction
0% ≤ mean % depletion ≤6.38%	No or minimal reactivity	Negative
6.38% < mean % depletion ≤22.62%	Low reactivity	Positive
22.62% < mean % depletion ≤42.47%	Moderate reactivity
42.47% < mean % depletion ≤100%	High reactivity

Table 2. The percentage peptide depletion (PPD) for lysine

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
WS-5 Test Article	37.49	37.26	-0.62 #	0.30	0.81
	37.41		-0.40 #
	36.93		0.89
Cinnamaldehyde Positive Control	17.63	37.26	52.68	50.35	4.61
	17.39		53.33
	20.48		45.03

#negative value was considered as “0” when calculating the mean

Table 3. The percentage peptide depletion (PPD) for cysteine

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
WS-5 Test Article	22.13	23.06	4.03	7.16	2.72
	20.99		8.98
	21.11		8.46
Cinnamaldehyde Positive Control	8.15	23.06	64.66	65.50	0.74
	7.89		65.78
	7.83		66.05

Applicant's summary and conclusion

Interpretation of results:

other: negative prediction for skin sensitisation

Conclusions:

The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay, performed according to OECD Guidelines for Testing of Chemicals Method 442C.

Executive summary:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article.

The test article, WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.

Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.

Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.

All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.

Conclusion:

The test article, WS-5, was considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay according to OECD TG 442 C.