Reaction mass of 2-methylbutyl salicylate and pentyl salicylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8 Oct 2018 to 12 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442c and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 5 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Conducted within the "spirit" of GLP (See Principles of method if other than guideline)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl salicylate

EC Number:

218-080-2

EC Name:

Pentyl salicylate

Cas Number:

2050-08-0

Molecular formula:

C12H16O3

IUPAC Name:

Pentyl 2-hydroxybenzoate

Constituent 2

Reference substance name:

2-methylbutyl salicylate

EC Number:

256-972-3

EC Name:

2-methylbutyl salicylate

Cas Number:

51115-63-0

Molecular formula:

C12H16O3

IUPAC Name:

2-methylbutyl 2-hydroxybenzoate

Test material form:

liquid

Specific details on test material used for the study:

Test material name (as stated in the report): AMYL SALICYLATE (pentyl 2-hydroxybenzoate )
Batch No.: VE00541093
Purity>=98%
Expiry date: 17 Apr 2019

In chemico test system

Details on the study design:

BASIS OF THE METHOD
Skin sensitizing chemicals have the ability to covalently modify skin proteins or to be biotically or abiotically activated to become protein-reactive. Chemical-modified proteins are recognized by the immune system as foreign and trigger a specific T-cell mediated immune response. A key step in the skin sensitization process is therefore the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. Based on this wellestablished toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins. Gerberick et al. [2, 4] therefore developed a peptide depletion assay using different heptapeptides (later coined the DPRA or ‘direct peptide reactivity assay’) to assess a chemicals ability to react with and deplete a test peptide. Depletion is measured as the loss of the peptide signal as determined by HPLC-UV.

EXPERIMENTAL DESCRIPTION
Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

HPLC-Conditions:
▪ LC-System: Agilent 1100 Series (quat. low pressure gradient pump)
▪ Column: Zorbax SB-C18, 3μm, 2.1mm x 100mm
▪ DAD-Detector: 220nm
▪ Inj.Vol.: 7μl, Temp.: 30°C, Flow: 0.35ml/min
▪ Mobile Phase: ACN + 0.085% TFA / H2O + 1% TFA
▪ Gradient: 0min: 10% ACN / 90% H2O
10min: 25% ACN / 75% H2O
11min: 90% ACN / 10% H2O
13min: 90% ACN / 10% H2O
13.5min - 20min: 10% ACN / 90% H2O (conditioning)

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control:
In each test Cinnamic aldehyde (EC: 203-213-9) (purity >99%) (supplied by Aldrich) is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

Prediction Model:
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.

The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals is described in Natsch, A., Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility. Givaudan Red Corner Report, RCR 153’453, 2015.

Results and discussion

Positive control results:

Cinnamic aldehyde

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Peptide Depletion Test sample, Average (%)

Parameter:

other: Cys-peptide depletion

Value:

1.1

Vehicle controls validity:

valid

Remarks:

Acetonitrile

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Peptide Depletion Test sample, Average (%)

Parameter:

other: Lys-peptide depletion

Value:

0

Vehicle controls validity:

valid

Remarks:

Acetonitrile

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

no indication of skin sensitisation

Remarks:

co-elution occured, Cysteine 1:10 prediction model was used to conclude

Other effects / acceptance of results:

Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (0.3 % and 2.2% SD, respectively).
The co-elution controls indicated no co-elution with an UV-absorbing component.

Any other information on results incl. tables

The test substance gave 1.1 %depletion of the Cys-peptide. With the Lys-peptide, co-elution was observed with a shoulder between RT 6 - 6.5 min and accurate quantification is thus not possible. Still the peak of the Lys peptide at the true retention time in the original height can be observed, indicating that no Lysine peptide depletion occurred. Manuel integration confirmed this (values given in brackets in Table 4), indicating negative depletion values due to partially overlapping peaks. Since quantification is not accurate, the Cysteine 1:10 prediction model was applied as indicated in the OECD test guideline. According this prediction model, the test substance is attributed to the “minimal” reactivity class (Cysteine-peptide depletion). In this Cysteine only model, chemicals are rated as sensitizers if they lead to >13.89% depletion of the Cysteine peptide.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
AMYL SALICYLATE was non-reactive and classified into the minimal reactivity class according to the Cysteine 1:10 prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Executive summary:

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance AMYL SALICYLATE was dissolved in acetonitrile and mixed with a Cysteine and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by AMYL SALICYLATE was determined by HPLC-UV.

AMYL SALICYLATE was non-reactive with the Cys-peptide. Co-elution occurred with the Lyspeptide. It was thus rated with the Cysteine 1:10 prediction model according to the guideline and classified into the minimal reactivity class. It is therefore considered a non-sensitizer according to the DPRA.