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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
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 4 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:
other: Direct peptide reactivity assay (DPRA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
Test material form:
liquid
Specific details on test material used for the study:
Trivial name : Baccartol Crude
Supplier : Givaudan Schweiz AG
Product code : 2376504
CAS number : 68916-62-1
Expiration date : 24.12.2016
Batch number : AS00187144
Physical form : Liquid
Concentrations tested : 12 binary dilutions from 2000 μM to 0.98 μM
Storage conditions : 4°C

In chemico test system

Details on study design:
The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test substances towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals [2-6] and it was found to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. The assay underwent validation at the European Center for the Validation of Alternative Methods (ECVAM) and an OECD guideline was adopted (OECD Test guideline 442c). The standard operating procedure (SOP) has been published by ECVAM. It was proposed by ECVAM to be to be used as part of an integrated approach for testing and assessment (IATA).

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 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 at the test facility is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility’.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Cys-peptide depletion
Value:
58.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: Lys-peptide depletion
Value:
0.69
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: Average depletion Cys-and Lys-peptide
Value:
29.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: Elution time Cys peptide
Value:
10.75
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: Elution time test substance Cys peptide run
Value:
15 - 16.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: 15 / 16.5 / 16.6
Parameter:
other: Elution time Lys peptide
Value:
8.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: Elution time test substance Lys peptide run
Value:
>= 14.6 - <= 16.3
Remarks on result:
other: 14.6 / 15 / 15.2 / 16.3

Any other information on results incl. tables

Test substance overall results:

   Average  Standard deviation
 Cys-peptide depletion  58.5  0.15
 Lys-peptide depletion  0.69  0.06
 Average depletion Cys-and Lys-peptide  29.6  
 Reactivity Class  MODERATE  
 Prediction  Sensitizer  
 Elution time Cys peptide  10.75  
 Elution time test substance Cys peptide run 15 / 15.5 / 16.6  
 Elution time Lys peptide 8.02  
 Elution time test substance Lys peptide run  14.6 / 15 / 15.2 / 16.3  

Applicant's summary and conclusion

Conclusions:
BACCARTOL CRUDE was peptide-reactive and classified into the moderate reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.
Executive summary:

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 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.