Tris(2-methylheptyl) 2-hydroxypropane-1,2,3-tricarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/9/2017 to 30/11/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (human Cell Line Activation Test (h-CLAT)

Version / remarks:

Adopted 29 July 2016.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

230-457-3

EC Name:

Tris(2-ethylhexyl) 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

7147-34-4

Molecular formula:

C30H56O7

Test material form:

liquid

Specific details on test material used for the study:

The test article, a clear liquid, was identified as Bernel Ester TCC and was received at Covance on 30 June 2017 as follows:
Test Article: Bernel Ester TCC
CAS Number: 16502-99-1
Storage: "15 to 25°C protected from light
Batch Number ESTS185/17, P7803
Retest Date 23-Jan-19
Purity 94%
A certificate of analysis for the test article was provided by the Sponsor. The solvent control was DMSO (at a concentration of 0.4% in culture medium). DMSO was supplied by Sigma Aldrich, Gillingham.

In chemico test system

Details on the study design:

Assay Acceptance Criteria

• The cell viabilities of medium and solvent control should be higher than 90%.
• In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
• For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%.
• For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

Negative Results

Negative results are acceptable only for test articles exhibiting a cell viability of less than 90% at 1.2 x CV75 (highest concentration). If the cell viability at 1.2 x CV75 is equal to or greater than 90% the negative results should be discarded and the dose selection should be refined by repeating the CV75 determination.
When the highest soluble concentration is used as the maximal test concentration of the test article, a negative result is acceptable even if the cell viability is above 90%.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Expression levels of CD86 and cell viability were analysed using flow cytometry. Cell viability >= 50 %

Parameter:

other: Relative Fluorescence Intensity - RFI (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Expression levels of CD54 and cell viability were analysed using flow cytometry. Cellviability >= 50 %

Parameter:

other: Relative Fluorescence Intensity - RFI (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

RESULTS

Dose Finding Assay

The cell viability results are given in the table below:

	Viability (%) at Concentration (µg/mL)
Run	0.781	1.562	3.125	6.25	12.5	25	50	100
1	99.1	98.6	98.8	99.0	98.9	99.0	99.3	99.0
2	99.1	98.8	99.2	99.3	99.0	99.2	99.0	98.8

No effect on viability was noted, therefore no CV75 value could be calculated.

CD86/CD54 Expression Results

Geometric mean fluorescence intensity (MFI) and viability results are given in the table below:

Experiment 1

	MFI (Geo Mean)			Corrected MFI		Viability
Concentration (ug/mL)	CD86	CD54	Isotype	CD86	CD54	CD86	CD54
27.9	1108	736	596	512	140	97.5	97.9
33.48	976	714	596	380	118	98.7	98.6
40.18	969	705	573	396	132	98.6	98.9
48.22	948	720	583	365	137	98.7	98.8
57.87	947	742	588	359	154	98.7	98.8
69.44	951	730	587	364	143	98.6	98.8
83.33	911	713	582	329	131	98.5	98.5
100	937	725	572	365	153	98.5	98.6
Solvent	1063	719	595	468	124	98.5	98.9
Positive control	2182	1141	717	1465	424	89.7	90.7

Experiment 2

	MFI (Geo Mean)			Corrected MFI		Viability
Concentration (ug/mL)	CD86	CD54	Isotype	CD86	CD54	CD86	CD54
27.9	1301	663	538	763	125	98.3	98.3
33.48	1276	661	536	740	125	98.4	98.8
40.18	1267	641	528	739	113	98.3	98.6
48.22	1322	658	539	783	119	98.3	98.3
57.87	1385	653	552	833	101	97.8	97.9
69.44	1336	662	557	779	105	98.4	98.7
83.33	1293	656	551	742	105	98.5	98.5
100	1392	672	550	842	122	98.3	98.8
Solvent	1282	675	571	711	104	98.7	99
Positive control	3097	2276	747	2350	1529	82.5	82.2

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (ug/mL)	RFI (CD86)		RFI (CD54)
Exp	Exp 1	Exp 2	Exp 1	Exp 2
27.9	109	107	113	120
33.48	81	104	95	120
40.18	85	104	106	109
48.22	78	110	110	114
57.87	77	117	124	97
69.44	78	110	115	101
83.33	70	104	106	101
100	78	118	123	117

Applicant's summary and conclusion

Interpretation of results:

other: The test article, Bernel Ester TCC, was considered to be negative in the human Cell Line Activation Test.

Conclusions:

CONCLUSION
The RFI of CD86 was <150% at all tested concentrations (with cell viability ≥50%) and the RFI of CD54 was <200% at all tested concentrations (with cell viability ≥50%) in both independent runs.
The test article, Bernel Ester TCC, was therefore considered to be negative in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of Bernel Ester TCC to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in anhydrous analytical grade dimethyl sulphoxide (DMSO) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).

Eight stock solutions were prepared by 1.2-fold serial dilutions using DMSO to give eight doses ranging from 0.335 x CV75 to 1.2 x CV75. These stock solutions were then diluted 250-fold (for DMSO) into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti- CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

No effect on viability was noted during the dose finding assay, therefore no CV75 value could be calculated.

The RFI of CD86 was <150% at all tested concentrations (with cell viability ≥50%) and the RFI of CD54 was <200% at all tested concentrations (with cell viability ≥50%) in both independent runs.

The test article, Bernel Ester TCC, was therefore considered to be negative in the human Cell Line Activation Test.