Pyrolysis Reaction Product of Pinane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-11-07 to 2019-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Pyrolysis Reaction Product of Pinane

EC Number:

947-964-7

Molecular formula:

n. a. for UVCB

IUPAC Name:

Pyrolysis Reaction Product of Pinane

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The U-SENS™ method is proposed to address this third key event by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and nonsensitisers.

Test System: U937 human monocytes.
Justification: Inducible CD86-expressing cells
Source: ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM.

Experimental design:
Plating of Cells
Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
Three valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 was rejected due to technical reasons and was repeated.

Treatment of Cells
Cells are treated for 45 ± 3 hours with the selected doses or controls (100 μL). The test item was in the first experiment evaluated up to 200 μg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 μg/mL.
In the second and third experiments cells were treated with 8 selected doses of the test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second and third experiment were as follows:
Second experiment: 90, 70, 50, 40, 30, 25, 20 and 15 μg/mL
Third experiment: 100, 90, 70, 50, 40, 30, 25 and 20 μg/mL
In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 μL.
After 45 ± 3 hours of exposure, wells were checked for precipitate.

Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 μL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 μL/well of the staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 μL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 μL of PBS.

Analysis of the cells was performed by flow cytometry.

ACCEPTABILITY CRITERIA
- At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
- The validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
- No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected
%CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
- The positive control (TNBS) is considered valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
- Negative control LA is considered valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

ANALYSIS
CV70 calculations
When the CV70 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation is done as follows:
For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated.

EC150 calculations
When the EC150 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation was done as follows:
For each CD86 well culture, the percentage of induced CD86+ cells is calculated as: [absolute %CD86+ — absolute%IgG1+]
A stimulation index (S.I.) is calculated for each dose level.
The viability for each dose level are the mean of the IgG1 well and the CD86 well. The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50% of CD86+ cells over the vehicle control) was calculated.

INTERPRETATION
- For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
- The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 X Median S.I. ≥ 150%).
- In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
- An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N) (Figure 1). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
- An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P) (Figure 1). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
- There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: S.I. of CD86 [%]

Value:

150

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: In the first experiment, the S.I. of CD86 is higher or equal to 150% at the highest noncytotoxic concentration only (50 µg/mL). This run is therefore considered ‘No Conclusion’ and two subsequent experiments were performed.

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: S.I. of CD86 [%]

Value:

150

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the S.I. of CD86 is higher or equal to 150% at the highest noncytotoxic concentration

Results 3

Key result

Run / experiment:

other: 3

Parameter:

other: the S.I. of CD86 [%]

Value:

150

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the S.I. of CD86 is higher or equal to 150% at all tested concentrations

Other effects / acceptance of results:

All three experiments passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100%, 98% and 99% in experiment 1, 2 and 3, respectively).
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in experiment 1 and 99% in experiment 2 and 3).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in all three experiments.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in all experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in all experiments.
- No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.
In all experiments the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Experiment 1

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 78 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 22 μg/mL.

-The test item showed colour interference at 100 and 200 μg/mL.

-The positive control (TNBS) showed a S.I. ≥508% in all wells and was non-cytotoxic atall concentrations (cell viability ≥70%). The negative control (LA) showed a S.I.≤97% in all wells and was non-cytotoxic at all concentrations (cell viability ≥70%).

 

Experiment 2

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 86 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 63 μg/mL.

-The test item showed no colour interference.

-The positive control (TNBS) showed a S.I. ≥591% in all wells and was non-cytotoxic atall concentrations (cell viability ≥ 70%).The negative control (LA) showed a S.I.≤109% in all wells and was non-cytotoxic at all concentrations (cell viability≥ 70%).

 

Experiment 3

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 61 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is < 20 μg/mL.

-The test item showed colour interference at 70 and 90 μg/mL.

- The positive control (TNBS) showed aS.I. ≥609% in all wells and was non-cytotoxic at all concentrations(cell viability ≥70%). The negative control (LA) showed a S.I.≤100% in all wells and was non-cytotoxic at all concentrations(cell viability ≥70%).

 

Table 1

Overview EC150 and CV70 Values

	EC150 (μg/mL)	CV70 (μg/mL)
Test item Experiment 1	22	78
Test item Experiment 2	63	86
Test item Experiment 3	< 20	61

 

Applicant's summary and conclusion

Interpretation of results:

other: positive, to be used as part of WoE-conclusion

Conclusions:

The test substance showed a positive result (potential to increase the expression levels of CD86 cell surface marker in the U937 cell line)

Executive summary:

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The test item was dissolved in dimethyl sulfoxide at 50 mg/mL, 22.5 mg/mL and 25 mg/mL in experiment 1, 2 and 3, respectively. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200μg/mL). In the second and third experiment, a narrower dose-response analysis was performed to investigate the increase in expression of experiment 1 up to 90 and 100 μg/mL (experiment 2 and 3, respectively) in more detail. No precipitate was observed at any dose level tested. Three independent experiments were performed. In the first experiment the test item showed toxicity (CV70 value of 78 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of 22 μg/mL) was measured. The individual run conclusion is considered No Conclusion, since the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. In the second experiment the test item showed toxicity (CV70 value of 86 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of 63 μg/mL) was measured. The individual run conclusion is considered Positive. An additional third experiment was performed, since the first two experiments were not concordant. In the third experiment the test item showed toxicity (CV70 value of 61 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of < 20 μg/mL) was measured. The individual run conclusion is considered Positive. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, the test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.