Cymbopogon winterianus, ext., reaction products with acetone

Skin sensitisation

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

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

Test material information

Constituent 1

Reference substance name:

Citronellol

EC Number:

203-375-0

EC Name:

Citronellol

Cas Number:

106-22-9

Molecular formula:

C10H20O

IUPAC Name:

3,7-dimethyloct-6-en-1-ol

Constituent 2

Reference substance name:

Geraniol

EC Number:

203-377-1

EC Name:

Geraniol

Cas Number:

106-24-1

Molecular formula:

C10H18O

IUPAC Name:

(E)-3,7-dimethylocta-2,6-dien-1-ol

Constituent 3

Reference substance name:

(E)-6,10-dimethylundeca-3,9-dien-2-one

Cas Number:

19870-49-6

Molecular formula:

C12H20O

IUPAC Name:

(E)-6,10-dimethylundeca-3,9-dien-2-one

Constituent 4

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene

Constituent 5

Reference substance name:

Linalool

EC Number:

201-134-4

EC Name:

Linalool

Cas Number:

78-70-6

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-1,6-dien-3-ol

Constituent 6

Reference substance name:

(±)-(1α,2β,5α)-5-methyl-2-(1-methylvinyl)cyclohexan-1-ol

EC Number:

256-557-7

EC Name:

(±)-(1α,2β,5α)-5-methyl-2-(1-methylvinyl)cyclohexan-1-ol

Cas Number:

50373-36-9

Molecular formula:

C10H18O

IUPAC Name:

rel-(1R,2S,5R)-5-methyl-2-(prop-1-en-2-yl)cyclohexanol

Constituent 7

Reference substance name:

(1α,2β,4β)-1-methyl-2,4-bis(methylvinyl)-1-vinylcyclohexane

EC Number:

251-713-0

EC Name:

(1α,2β,4β)-1-methyl-2,4-bis(methylvinyl)-1-vinylcyclohexane

Cas Number:

33880-83-0

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,2R,4S)-1-methyl-2,4-di(prop-1-en-2-yl)-1-vinylcyclohexane

Constituent 8

Reference substance name:

(1E,6E)-8-isopropyl-1-methyl-5-methylenecyclodeca-1,6-diene

Cas Number:

105453-16-5

Molecular formula:

C15H24

IUPAC Name:

(1E,6E)-8-isopropyl-1-methyl-5-methylenecyclodeca-1,6-diene

Constituent 9

Reference substance name:

rel-(1R,4aR,8aS)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene

Cas Number:

31983-22-9

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,4aR,8aS)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene

Constituent 10

Reference substance name:

rel-(1R,4aS,8aS)-1-isopropyl-7-methyl-4-methylene-1,2,3,4,4a,5,6,8aoctahydronaphthalene

Cas Number:

39029-41-9

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,4aS,8aS)-1-isopropyl-7-methyl-4-methylene-1,2,3,4,4a,5,6,8aoctahydronaphthalene

Constituent 11

Reference substance name:

rel-(1R,8aS)-1-isopropyl-4,7-dimethyl-1,2,3,5,6,8ahexahydronaphthalene

Cas Number:

60305-17-1

Molecular formula:

C15H24

IUPAC Name:

rel-(1R,8aS)-1-isopropyl-4,7-dimethyl-1,2,3,5,6,8ahexahydronaphthalene

Constituent 12

Reference substance name:

rel-2-((1R,3S,4S)-4-methyl-3-(prop-1-en-2-yl)-4-vinylcyclohexyl)propan-2-ol

Cas Number:

23811-48-5

Molecular formula:

C15H26O

IUPAC Name:

rel-2-((1R,3S,4S)-4-methyl-3-(prop-1-en-2-yl)-4-vinylcyclohexyl)propan-2-ol

Constituent 13

Reference substance name:

5,9-dimethyl-2-(6-methylhept-5-en-2-yl)deca-2,8-dienal

Cas Number:

82451-80-7

Molecular formula:

C20H34O

IUPAC Name:

5,9-dimethyl-2-(6-methylhept-5-en-2-yl)deca-2,8-dienal

Constituent 14

Reference substance name:

Likely oxygenated constituent

IUPAC Name:

Likely oxygenated constituent

Constituent 15

Reference substance name:

Unknown Impurities

IUPAC Name:

Unknown Impurities

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 the 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

Results

Results 1

Parameter:

other: Cys-peptide depletion

Value:

58.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Parameter:

other: Lys-peptide depletion

Value:

0.69

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Parameter:

other: Average depletion Cys-and Lys-peptide

Value:

29.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 4

Parameter:

other: Elution time Cys peptide

Value:

10.75

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Parameter:

other: Elution time test substance Cys peptide run

Value:

15

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: 15 / 16.5 / 16.6

Results 6

Parameter:

other: Elution time Lys peptide

Value:

8.02

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 7

Parameter:

other: Elution time test substance Lys peptide run

Value:

14.6

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.