Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 March 2017 and 08 Apr 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:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate

IUPAC Name:

Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1- ylidene]methyl}amino)benzoate

Test material form:

liquid

In chemico test system

Details on the study design:

Test Article Preparation
The test article was prepared at a 100 mM concentration in an appropriate solvent. Calculations using the molecular weight and purity of the test article were performed to determine the appropriate amount of test article to weigh out in order to achieve approximately 3 mL of the 100 mM sample.

The test article was weighed into a prelabeled glass vial and stored at room temperature. The test article was not dissolved in the solvent until immediately before mixing with the peptides.

Test Article Solubility TestA solubility test was performed for the test article in order to determine an appropriate solvent that completely dissolved the test article at a 100 mM concentration. The test article, Lyrame, was found to be soluble in acetonitrile with vortexing for approximately 1 minute.

Peptide Preparation
Custom synthetic peptides of cysteine or lysine (containing phenylalanine to aid in detection) were used in this assay. The purity of each peptide was at least 90%. Peptide samples were newly prepared for each sample set, and a single preparation of the peptide was used throughout the sample set. The cysteine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.6 phosphate buffer. The lysine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 10.1 ammonium acetate buffer. The peptide solutions were gently mixed on the shaker.

Peptide Standards
A set of serially diluted standards were prepared for each peptide. These standards were prepared by diluting the peptide solutions in dilution buffer (20% acetonitrile in either phosphate or ammonium acetate buffer). Six standards were prepared at concentrations of 0.534- 0.0167 mM. A seventh standard was prepared containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the appropriate prelabeled autosampler vials.

Controls
The positive control used in this assay was cinnamic aldehyde prepared at a concentration of 100 mM. The positive control was reacted with the peptides in the same fashion as the test article. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). Solvent controls were also prepared for the solvent used in the assay(if different than acetonitrile). These controls consist of the solvent (acetonitrile) reacted with the peptide in the absence of test article. A coelution control was also prepared for the test article. The coelution control consisted of the test article without the peptide. The purpose of the coelution control was to determine if the test article elution from the HPLC column overlapped with the peptide elution.

HPLC Set-up and Operation
The separation module used in this assay was a Waters 2690/5 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test article and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The dimensions of the column used were 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least two hours before the start of the assay. To prime the column, equal parts of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.08% trifluoroacetic acid in HPLC grade acetonitrile) were passed through the column.

Once the column was equilibrated and the samples were prepared, the autosampler vials were placed into the designated locations of the separations module carousels. The samples were incubated in the dark at room temperature for 24± 2 hours.

A gradient elution was used in this assay. The mobile phase changed from 10-25% acetonitrile over a 10 minute period to allow for optimal separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections.

The Empower PDA software (Version 3) was used to convert the absorbance data from the UV detector into chromatograms of intensity versus retention time for each sample and control. At the end of the run, each chromatogram was integrated in order for the software to calculate the area under the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible to determine which peaks in the chromatograms represented the peptides and use the areas under those peaks for the subsequent calculations.

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

other: Cysteine and Lysine

Parameter:

other: Mean Depletion (%)

Value:

13.55

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Solubility Assessment
The solubility of Lilial-Me Anthranilate in acetonitrile at a nominal concentration of 100 mM was confirmed by visual inspection.

Reactivity Assessment
All analytical acceptance criteria for each analytical run were met.
Only the cysteine result is reported by the application of the cysteine 1:10 reactivity prediction model (co eluting peaks). Based on this model, reactivity is classed as “no to minimal”, hence the DPRA prediction is negative.

Any other information on results incl. tables

DPRA Results

 

IIVS Test Article Number	Sponsor’s Designation	% Mean Peptide Depletion		% Mean Peptide Depletion of Cysteine and Lysine	Reactivity (Cysteine and Lysine)	Potential Sensitizer?
		Cysteine	Lysine
17AA44	Lyrame	17.02	10.07	13.55	Low	Yes
Positive Control	Cinnamic Aldehyde	74.78	61.55

Applicant's summary and conclusion

Interpretation of results:

other: “low reactivity” and thus is predicted to be positive by DPRA for skin sensitization potential.

Conclusions:

Solutions of Lyrame were successfully analyzed by the validated DPRA analytical method in the Cysteine or Lysine containing synthetic peptides with all analytical acceptance criteria of the test being met.

The Mean Peptide Depletion of Cysteine and Lysine in the presence of Lyrame was 13.55% and from this result a DPRA prediction can be made. The reactivity of Lyrame with Cysteine and Lysine is classed as “low reactivity” and thus is predicted to be positive.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Lyrame.

Solutions of Lyrame were successfully analyzed by the validated DPRA analytical method in the Cysteine or Lysine containing synthetic peptides with all analytical acceptance criteria of the test being met.

The Mean Peptide Depletion of Cysteine and Lysine in the presence of Lyrame was 13.55% and from this result a DPRA prediction can be made. The reactivity of Lyrame with Cysteine and Lysine is classed as “low reactivity” and thus is predicted to be positive.