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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:
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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

Resultsopen allclose all
Parameter:
other: Relative Fluorescence Intensity - RFI (%)
Run / experiment:
Expression levels of CD86 and cell viability were analysed using flow cytometry. Cell viability >= 50 %
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Relative Fluorescence Intensity - RFI (%)
Run / experiment:
Expression levels of CD54 and cell viability were analysed using flow cytometry. Cellviability >= 50 %
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.