Castor oil, dehydrated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 December 2012 until 14 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to respective guidelines and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline available

Guideline:

other: At the time of this report, no adopted national or international guidelines for the in vitro sensitisation test battery evaluation nor the individual tests were available. However, the individual studies were performed according published methodology.

Deviations:

not applicable

Principles of method if other than guideline:

Chemical reactivity has been shown to be well associated with the allergenic potency. Within this context measuring the amount of proteins with nucleophillic side chains sich as cysteine or lysine residues after incubation with putative allergens may serve as surrogate markers. Due to the complexity of the skin sensitisation process a single in vitro assay is not sufficient to address this endpoint. Therefore a combination of several methods addressing two majore steps of the sensitisation process: protein reactivity and activation of dendritic cells has been proposed in a strategy to assess the sensitising potential.
The test substance was incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration was determined by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220nm. The peptides are custom-made material containing phenylalanine to facilitate UV-detection and either cysteine or lysine as the reactive center. Based on the molecular weight of the test subtance and the peptides, the test substance should be incubated with the C-containing peptide in a ratio of 1:10 (0.5nM peptide, 5 nM test substance) and with the K-containing peptide in a ratio of 1:50 (0.5 nM peptide, 25nM test substance). However, as no molecular weight of the test substance was available a gravimetric procedure was applied. The test substance was dissolved in propanol at a concentration of 3.76% (corresponding to 100nM at a theoretical molecular weight of ca. 375 g/mol and a purity of 100%). Three samples of the test substance were incubated with each peptide in ratios of 1:5 (0.376 mg peptide, 1.88 mg test substance; for C-peptide0 or 1:24 (0.389 mg peptide, 9.4 mg test substance; for K-peptide) based on absolute mass. Additionally triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test
substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity. The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.
For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity.

GLP compliance:

yes

Type of study:

other: Reactivity of the test substance towards synthetic cysteine (C) or lysine (K)-containing peptides was evaluated.

Test material

Test material information

Constituent 1

Reference substance name:

Castor oil, dehydrated

EC Number:

264-705-7

EC Name:

Castor oil, dehydrated

Cas Number:

64147-40-6

Molecular formula:

Not applicable for this UVCB.

IUPAC Name:

Castor oil, dehydrated

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Castor oil, dehydrated
- Physical state: Liquid, yellowish, clear
- Lot/batch No.: 12.002.030
- Storage condition of test material: ambient room temperature, dry storage, under light exclusion
- Other:

In vivo test system

Test animals

Species:

other: not applicable

Strain:

other: Not applicable

Sex:

not specified

Details on test animals and environmental conditions:

Not applicable as it is an in vitro study

Study design: in vivo (non-LLNA)

Induction

Route:

other: Not applicable

Vehicle:

other: see below for information on vehicle

Challenge

Route:

other: Not applicable

Vehicle:

other: see below for information on vehicle

No. of animals per dose:

Not applicable

Details on study design:

Not applicable since this study is not a traditional test

Challenge controls:

Negative control (NC): vehicle control = propanol (PrOH). Acetonitrile (ACN) was performed as an additional vehicle control for the PC EGDMA.
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) (prepared as a 50 mM solution in propanol and inacetonitrile). As EGDMA may result in different peptide depletions when formulated in different vehicles the standard vehicle was used in addition to the vehicle used for test substance formulation.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Positive control substance(s):

yes

Remarks:

Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) (prepared as a 50 mM solution in propanol and inacetonitrile).

Results and discussion

Any other information on results incl. tables

Reaction with cysteine-peptide

Reaction with cysteine peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3	Mean	SD
NC: PrOH	724.5	715.5	709.2	0.492	0.486	0.482	0.487	0.005
Castor oil, dehydrated - 12/0806-1	715.8	outlier*	698.9	0.486	-	0.475	0.481	0.008
PC: EGDMA in PrOH	376.6	346.0	304.5	0.259	0.239	0.211	0.236	0.024
PC: EGDMA in ACN	383.4	349.6	340.5	0.264	0.241	0.235	0.247	0.015

 

Reaction with cysteine peptide	Peptide depletion %
	sample 1	sample 2	sample 3	Mean	SD
NC: PrOH	-1.1	0.1	1.0	0.0	1.1
Castor oil, dehydrated - 12/0806-1	0.1	-	2.4	1.2	1.6
PC: EGDMA in PrOH	46.7	50.9	56.7	51.5	5.0
PC: EGDMA in ACN	45.6	50.3	51.5	49.2	3.1

 

 

 

Reaction with cysteine peptide	peak area [mAU*s] at 258 nm			Area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
NC: PrOH	18.7	20.3	20.3	38.7	35.2	35.0
Castor oil, dehydrated - 12/0806-1	20.1	-	19.8	35.7	-	35.3
PC: EGDMA in PrOH	10.4	9.2	8.2	36.1	37.6	37.1
PC: EGDMA in ACN	10.5	9.7	9.4	36.4	36.1	36.0

 

The peptide concentration and peptide depletion of the PC: EGDMA in ACN was calculated relative to the vehicle acetonitrile. The NC acetonitrile had a mean cysteine-peptide concentration of 0.485 mM (single data not shown).

*Sample 2 of the test substance was considered to be an outlier (peak area at 220 nm: 575.2) probably caused by an error during sample preparation and was hence not used for calculations.      

 

Reaction with lysine-peptide

Reaction with lysine peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3	Mean	SD
NC: PrOH	662.6	662.7	661.2	0.495	0.495	0.494	0.495	0.001
Castor oil, dehydrated - 12/0806-1	673.3	679.7	674.8	0.503	0.508	0.504	0.505	0.002
PC: EGDMA in PrOH	581.6	577.9	566.3	0.434	0.432	0.423	0.430	0.006
PC: EGDMA in ACN	587.6	586.8	580.9	0.439	0.438	0.434	0.437	0.003

 

Reaction with lysine peptide	Peptide depletion %
	sample 1	sample 2	sample 3	Mean	SD
NC: PrOH	-0.1	-0.1	0.1	0.0	0.1
Castor oil, dehydrated - 12/0806-1	-1.8	-2.7	-1.9	-2.1	0.5
PC: EGDMA in PrOH	12.2	12.8	14.5	13.2	1.2
PC: EGDMA in ACN	12.8	12.9	13.8	13.1	0.5

 

 

 

Reaction with lysine peptide	peak area [mAU*s] at 258 nm			Area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
NC: PrOH	19.0	18.9	18.4	34.9	35.1	36.0
Castor oil, dehydrated - 12/0806-1	19.4	19.5	18.7	34.7	34.9	36.0
PC: EGDMA in PrOH	16.7	16.4	16.7	34.8	35.2	33.8
PC: EGDMA in ACN	16.8	16.9	16.9	35.0	34.7	34.3

 

The peptide concentration and peptide depletion of the PC: EGDMA in Can was calculated relative to the vehicle acetonitrile. The NC acetonitrile had a mean lysine-peptide concentration of 0.503 mM (single data not shown).

Additional observations

The test substance was solved in propanol. However, when mixed with the peptide stock solutions the samples became opaque. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples but the samples were emulsions, still. Thus the samples were centrifuged prior to HPLC analysis. No co-elution of test substance and peptide occurred as demonstrated by the area ratio 220/258 and the co-elution control.

 

Mean peptide depletion

	Cysteine-peptide		Lysine-peptide		Mean of both depletions %
	Mean depletion %	SD	Mean depletion %	SD
Castor oil, dehydrated - 12/0806-1	1.2	1.6	-2.1	0.5	0.6
PC: EGDMA in PrOH	51.5	5.0	13.2	1.2	32.3
PC: EGDMA in ACN	49.2	3.1	13.1	0.5	31.2

 

Chemical reactivity was determined by mean peptide depletion and was rated as high,

moderate, low, or minimal³:

 

Mean peptide

depletion [%]                 Reactivity

 

> 42.47                                high reactivity

> 22.62 < 42.47                moderate reactivity

> 6.38 < 22.62                   low reactivity

< 6.38                               minimal reactivity

³According to the classification tree model described by Gerberick et al. for substances with available molecular weight highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer, and a test substance of minimal reactivity (mean peptide depletion < 6.38 %) a non-sensitizer.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance shows a minimal chemical reactivity in the DPRA assay.

Executive summary:

In the Direct Peptide Reactivity Assay (DPRA), the reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated (Kolle, 2013a). For this purpose, the substance was incubated with synthetic peptides for 24 h at room temperature and the remaining non-depleted peptide concentration was determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was solved in propanol and incubated in ratios of 1:5 for C-peptide or 1:24 for K-peptide based on absolute mass. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analysed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity. The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance, the mean peptide depletion as average of C- and K-peptide depletion was calculated and used for evaluation of the chemical reactivity. Based on the observed results and applying the prediction model proposed in Gerbericket al.(2007), it was concluded that castor oil, dehydrated showed minimal chemical reactivity in the DPRA under the chosen test conditions (Kolle, S, N, 2013).