Reaction product of castor oil with glycerol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 - 20 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No historical control data range reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

adopted in 2017

Deviations:

yes

Remarks:

Adapted to animal product-free conditions by the test facility and validated in-house to determine denditric cell activation.

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines & Healthcare products Regulatory Agency, Department of Health of the Government of the United Kingdom

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of castor oil with glycerol

EC Number:

949-117-7

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Reaction product of castor oil with glycerol

Test material form:

liquid

In vitro test system

Details on the study design:

TEST METHOD:
The h-CLAT system has been modified to remove all animal-derived components, this involves the use of Human Serum instead of Foetal Calf Serum for cell culture, Human Serum Albumin instead of Bovine Serum Albumin in the Flow Cytometry staining buffer and use of custom anti-CD54 and anti-CD86 antibodies from a non-animal source (HuCAL).

The in vitro human Cell Line Activation Test (h-CLAT) is an alternative testing method for the evaluation of the skin sensitisation potential of a test substance. It quantifies phenotypic changes, such as cell surface marker expression in cell lines following 24 h treatment with chemicals. The human leukemia cell line THP-1 is used as surrogate for human myeloic dendritic cells, which show enhanced CD86 and CD54 surface protein expression when treated with sensitiziers.
The dose for the h-CLAT assay was determined in two preliminary cytotoxicity test, yielding 75% cell viability (CV75). For the main assay, THP-1 cells were incubated for 24 ± 1 h at 37 °C with the test substance, as well as the negative and positive controls. Changes of CD86 and CD54 expression were analysed by flow cytometry, using fluorescently labelled antibodies against the two surface proteins. Relative fluorescence intensities (RFI) compared to solvent controls are calculated and used in a prediction model to discriminate between sensitising and non-sensitising compounds.

TESTS SUBSTANCE PREPARATION:
The test substance was dissolved in complete culture medium.

CONCENTRATIONS:
Pre-experimental (CV75 determination): 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL
Main experiment (h-CLAT): 1137.82, 948.18, 790.15, 658.46, 548.72, 457.26, 381.05 and 317.54 µg/mL (based on the results obtained in the pre-experimental dose-finding study)

VEHICLE CONTROL: Complete Roswell Park Memorial Institute (RPMI) culture medium containing 10% human serum and 0.05 mM 2-mercaptoethanol
POSITIVE CONTROL CV75 determination: 2,4-Dinitrochlorobenzene (DNCB, Supplier: Sigma-Aldrich, Batch no. BCBP8259V, Expiry date: 06 Feb 2021), 8 µg/mL in dimethyl sulfoxide (DMSO)
POSITIVE CONTROL CD54 and CD86 expression: Nickel sulphate (supplier: Sigma-Aldrich, Batch no. SZBF2960V, Expiry date: 15 June 2021), 100 µg/mL in RPMI medium

TEST CELL LINE: THP-1 cells
- Source: ATCC (via LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK), Cat. #TIB-202

CELL CULTURE CONDITIONS:
- Type and identity of media: RPMI supplemented with 10% Human Serum and 0.05 mM 2-mercaptoethanol

EXPOSURE CONDITIONS:
- Method of application: in medium
- Exposure duration: 24 ± 0.5 h

NUMBER OF REPLICATES: Each concentration was tested in two independent runs

DETERMINATION OF CYTOTOXICITY:
- Method: Propidium iodide, 24 ± 0.5 h exposure with test item, two independent experiments
- Determination of cell viability (= relative absorbance) for calculation of the CV75, which corresponds to the concentration needed to reduce the relative absorbance to 75% of the solvent control.

DETERMINATION OF FLUORESCENCE:
- Flow cytometry
- Antibodies: fluorochrome-tagged CD86 and CD54

Results and discussion

Positive control results:

RFI, first experiment:
100 µg/mL: CD54 = 212% (93.69% viability), CD86 = 152% (93.04% viability)

RFI, second experiment:
100 µg/mL: CD54 = 377% (88.95% viability), CD86 = 210% (86.58% viability)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

in 2/2 independent experiments

Results 2

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD86 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

in 2/2 independent experiments

Other effects / acceptance of results:

- Acceptance criteria met for CV75 determination: Yes, cell viability is ≥ 75% at the lowest dose and the highest test item concentration produces cytotoxicity (< 90% cell viability)

- Acceptance criteria met for negative control: yes, medium and solvent control RFI values do not exceed the positive criteria CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 90%

- Acceptance criteria met for positive control: yes, RFI values CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%

Any other information on results incl. tables

Table 1: Results of the h-CLAT test, first experiment

Test Item Dose (µg/ml)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
1137.82	24.66	23.94	21.55	23.39	380	438
948.18	45.06	45.75	39.07	43.29	204	168
790.15	93.75	93.08	92.37	93.06	57	86
658.46	96.05	95.57	94.85	95.49	72	86
548.72	97.30	96.69	96.55	96.85	77	106
457.26	97.12	96.53	96.60	96.75	81	109
381.05	97.40	96.42	96.72	96.85	89	93
317.54	97.28	96.83	96.26	96.79	79	102

The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at 1137.82 and 948.18 µg/mL in the first experiment 1 (pink cells). However, as the viability was < 50% (grey cells) at both doses this is considered negative. The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at 1137.82 and 948.18 µg/mL in the first experiment (green cells). However, the viability was < 50% at both doses (grey cells) so this is considered negative.

Table 2: Results of the h-CLAT test, second experiment

Test Item Dose (µg/ml)	Cell Viability (%)			Average Cell Viability (%)	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
1137.82	20.09	20.41	22.26	20.92	577	298
948.18	49.87	51.12	51.85	50.95	136	65
790.15	93.07	93.49	92.70	93.09	90	40
658.46	93.55	93.05	94.38	93.66	128	85
548.72	94.46	95.05	95.88	95.13	117	92
457.26	95.15	95.78	96.28	95.74	113	88
381.05	95.03	95.84	95.86	95.58	114	109
317.54	94.30	95.27	95.90	95.16	83	60

The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at 1137.82 µg/mL in the second experiment (pink cell). However, as the viability was < 50% this is considered negative (grey cell). The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at 1137.82 µg/mL in the second experiment (green cell). However, the viability was < 50% so this is considered negative (grey cell).

Table 3: The CV75 value derived from two independent experiments

CV75 (Rep1)	CV75 (Rep2)	Average CV75 (µg/ml)
1419.15	477.22	948.18

Table: 4: Proficiency data generated using the XCellR8 animal product-free adaptation of the h-CLAT method

Test Item	OECD h-CLAT Classification	OECD EC200 range (µg/ml)	OECD EC150 range (µg/ml)	XCellR8 h-CLAT Classification	XCellR8 EC200 (µg/ml)	XCellR8 EC150 (µg/ml)
2,4-dinitrochlorobenzene	Sensitiser	0.5-15	0.5-10	Sensitiser	5	4
4-Phenylenediamine	Sensitiser	Neg or > 1.5	< 40	Sensitiser	13	12
Nickel sulfate	Sensitiser	10-100	< 100	Sensitiser	48	44
2-Mercaptobenzothiazole	Sensitiser	10-140	Neg or > 10	Sensitiser	103	63
R(+)-Limonene	Sensitiser	< 250	Neg or > 5	Sensitiser	78	41
Imidazolidinyl urea	Sensitiser	20-75	20-90	Sensitiser	39	40
Isopropanol	Non-sensitiser	Neg (> 5000)	Neg (> 5000)	Non-sensitiser	Neg (> 5000)	Neg (> 5000)
Glycerol	Non-sensitiser	Neg (> 5000)	Neg (> 5000)	Non-sensitiser	Neg (> 5000)	Neg (> 5000)
Lactic acid	Non-sensitiser	Neg (> 5000)	Neg (> 5000)	Non-sensitiser	Neg (> 1856)*	Neg (> 1856)*
4-Aminobenzoic acid	Non-sensitiser	Neg (> 1000)	Neg (> 1000)	Non-sensitiser	Neg (> 600)**	Neg (> 600)**
The h-CLAT system has been modified to remove all animal derived components, this involves the use of Human Serum instead of Foetal Calf Serum for cell culture, Human Serum Albumin instead of Bovine Serum Albumin in the Flow Cytometry staining buffer and use of custom anti-CD54 and anti-CD86 antibodies from a non-animal source (HuCAL).*Due to the experimental CV75 derived (1546 µg/ml) the highest dose tested for Lactic Acid was 1856 µg/ml. **Due to solubility limitations the highest dose tested for 4-Aminobenzoic acid was 600 µg/ml.

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event 'activation of dendritic cells'

Conclusions:

There is regulatory acceptance in the EU for the application of the h-CLAT test method to address key event 3, monocytic / dendritic cell response, in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance did not induce dendritic cell activation. However, test substances with a log Kow greater than 3.5 tend to produce false negative results in the h-CLAT test. Negative results obtained with test substances with a log Kow greater than 3.5 should not be considered. Since the log Kow values of the different constituents of the test substance range from -4.09 to 3.94 and are, therefore, partly outside the applicability domain of the test, the negative result is not considered for the hazard assessment of the registered substance. Further evaluation and/or data generation is required.

Executive summary:

The available data on skin sensitisation indicate a positive sensitising potential based on the key event 'inflammatory response to keratinocytes' (OECD 442D) (key event 2 of the adverse outcome pathway of skin sensitisation), a negative sensitising potential based on the key event 'dendritic cell activation' in the h-CLAT test (OECD 442E) and a positive sensitising potential in the GARD (Genomic Allergen Rapid Detection)skin (scientifically validated, approved for the OECD Test Guideline Program (TGP No. 4.106), both assays investigate key event 3 of the adverse outcome pathway of skin sensitisation). However, test substances with a log Kow > 3.5 tend to produce false negative results in the h-CLAT test. Negative results obtained with substances with a log Kow > 3.5 should not be considered in the hazard assessment of those substances. Since the log Kow values of some of the constituents of the registered substance are > 3.5, the negative result is not considered. Only the positive result of the GARDskin assay is used to conclude on key event 3 of the adverse outcome pathway of skin sensitisation.

There is regulatory acceptance in the EU that the skin sensitisation potential of a substance can be reliably assessed based on a '2 out of 3' rule, i.e. the results of adequate studies on 2 key events can be used to conclude on the hazard assessment and classification and labelling. Since there are positive results for 2 key events ('inflammatory response to keratinocytes' and 'dendritic cell activation'), reaction product of castor oil with glycerol meets the classification critera of Regulation (EC) No. 1272/2008 (CLP) and is classified as Skin Sens. 1 (H317).