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Administrative data

Description of key information

The test item was evaluated for skin sensitisation using the DPRA (OECD 442C), KeratinoSens (OECD 442D) and U-SENS (OECD 442E). A negative response was obtaind in the DPRA and keratinoSens. A positive response was obtained in an U-SENS study. According to the "2-out-of-3" prediction model, the test item can be considered as a non-sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 January 2018 - 24 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
GLP compliance:
yes
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.
Details on study design:
TEST ITEM PREPERATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v). Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 49.28 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1384 μL isopropanol after vortex mixing and 1 minute of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler, in the dark, and incubated at 25 ± 2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018


DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see Table 3 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Key result
Parameter:
other: SPCC mean percentage
Run / experiment:
Cysteine Reactivity Assay
Value:
3.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 2.3%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 76.5% ± 1.1%
Remarks on result:
other: SD: 2.8%
Key result
Parameter:
other: SPCL mean percentage
Run / experiment:
Lysine Reactivity Assay
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 1.7%
Positive controls validity:
valid
Remarks:
mean percentage SPCL: 55.3% ± 0.9%
Remarks on result:
other: SD: 0%
Other effects / acceptance of results:
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, phase separation (formation of oily droplets in the solution) was observed.
See table 4 and 5 in " any other information on results incl. table" for results and acceptability of DPRA

Table 4: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay 

Lysine reactivity assay

Acceptability criteria

Results for

SPCC

Acceptability criteria

Results for

SPCL

Correlation coefficient (r2) standard calibration curve 

>0.99

0.993

>0.99

0.997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05 

0.525 ± 0.004

0.50 ± 0.05

0.491 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.518 ± 0.003

0.50 ± 0.05

0.482 ± 0.006

Mean peptide concentration RC-Cisopropanolsamples (mM)

0.50 ± 0.05

0.501 ± 0.001

0.50 ± 0.05

0.486 ± 0.013

CV (%) for RC samples B and C

<15.0

2.3

<15.0

1.7

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

76.5

40.2-69.0

55.3

SD of peptide depletion cinnamic aldehyde (%)

<14.9

1.1

<11.6

0.9

SD of peptide depletion for the test item (%)

<14.9

2.8

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 5: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item 

SPCC depletion 

SPCL depletion

Mean of

SPCC and

SPCL

depletion

DPRA prediction and     reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

Hostacor ITD

3.3%

±2.8%

0.0%

±0.0%

1.6%

Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
Hostacor ITD was negative in the DPRA and was classified in the “No or minimal reactivity” class when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation (formation of oily droplets in the solution) was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of Hostacor ITD towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers.

Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model.

Isopropanol was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.  Cinnamic aldehyde was used as a positive control. The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test substance, were within the acceptability criteria for the DPRA assay. Therefore, the study was considered to be valid.

In the cysteine reactivity assay the test item showed 3.3% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.6% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

However, since phase separation (formation of oily droplets in the solution) was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 February 2018 - 02 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The KeratinoSensTM assay is recommended in international guidelines (e.g. OECD) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
Details on study design:
TEST ITEM PREPARATION
Based on a solubility test, a concentration of 2000 μM was selected as highest concentration for the main assay. This dose was the highest dose required in the current guideline.

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

Test item concentrations were used within 3 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of a solvent control of 1% DSMO were tested
- Blank control: on each plate three bank wells were tested (no cells and no treatment)

TEST DESIGN
- Test system: The KeratinoSens™ cell line, having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+16 in experiment 1 and P+18 in experiment 2.

- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.

- Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in a microplate reader to assess the quantity of luciferase (integration time two seconds).

- Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue tetrazolium bromide and subsequently cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with a microplate reader.

DATA ANALYSIS: according to guideline

Interpretation of results as positive:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 μg/mL should be considered as inconclusive.

Acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
•The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.28 and the EC1.5 120 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.04 and the EC1.5 54 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
0.97
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: could not be calculated
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
0.82
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: could not be calculated
Other effects / acceptance of results:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (120 μM and 54 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.28-fold and 2.04-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.1% and 8.9% in experiment 1 and 2, respectively).


CYTOTOXICITY:
The test item showed toxicity in both experiments

Experiment 1
IC30: 21 µM
IC50: 24 µM

Experiment 2
IC30: 1.8 µM
IC50: 18 µM

PRECIPITATION:
Precipitation was observed at the start of the incubation period at test concentrations of 500, 1000 and 2000 μM in both experiments. At the end of the incubation period precipitation was observed at test concentrations of 2000 μM in experiment 1 and 1000 and 2000 μM in experiment 2.
Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
Phosphoric acid, isotridecyl ester (Hostacor ITD) is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.
Executive summary:

A KeratinoSens(TM) assay was performed with Phosphoric acid, isotridecyl ester (Hostacor ITD) according to OECD 442D and GLP principles.

The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item precipitated at the dose level of 2000 μM in experiment 1 and at dose levels of 1000 and 2000 μM in experiment 2. Two independent experiments were performed. Both experiments passed the acceptance criteria.

The test item showed toxicity (IC30 values of 21 μM and 1.8 μM and IC50 values of 24 μM and 18 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.97-fold and 0.82-fold in experiment 1 and 2 respectively.

In conclusion, Phosphoric acid, isotridecyl ester (Hostacor ITD) is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 April 2018 - 27 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
One of the validated in vitro skin sensitization tests is the U-SENS assay, which is recommended in international guideline (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
Qualifier:
according to
Guideline:
other: OECD 442E Annex II, In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™)
Version / remarks:
9 October 2017
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The U-SENS is recommended in international guidelines (e.g. OECD) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.
Details on study design:
TEST ITEM PREPARATION
Based on a solubility test, DMSO was selected as solvent for the main assay. This solvent was the first choice as is stated in the current guideline.
In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 μg/mL in the 96-well plate (final concentration DMSO of 0.4%) in the first and second experiment. In the third and fourth experiment the stock was diluted to eight test concentrations (7.5, 10, 12.5, 15, 20, 30, 40 and 60 μg/mL and 10, 15, 20, 25, 30, 35, 40 and 50 μg/mL resp.) Test item concentrations were used within 2 hours after preparation.

CONTROL ITEMS ( all controls in triplicate)
- Positive control: 2,4,6-Trinitrobenzenesulfonic acid (50 μg/mL)
- Negative control: Lactic acid (200 μg/mL)
- Vehicle control: 0.4% Dimethyl sulfoxide in complete medium
- Blank control: Complete medium

TEST DESIGN
- Cells: U937 human monocytes.
- Source: ATCC (American Type Culture Collection, Virginia, USA).
Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times.
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 71 – 99 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 37.3°C).

- Plating of cells: Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0 x 1E5 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.

- 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 and including 200 μg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 μg/mL. In the second and third experiment cells were treated with six and eight selected doses of test item, respectively. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second, third and fourth experiment were 1.0, 10, 20, 50, 100 and 200 μg/mL, 7.5, 10, 12.5, 15, 20, 30, 40 and 60 μg/mL and 10, 15, 20, 25, 30, 35, 40 and 50 μg/mL.
Four valid experiments were conducted per test item. One experiment was rejected since the acceptability was not met.

- Antibody staining:
FITC-conjugated antibodies were 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). 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.

- Flow cytometry method: Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used. Color interference was evaluated.

- Color interference on IgG1 analysis: There is colour interference in the IgG1 evaluation when the X Median of the FITC fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

DATA ANALYSIS: according to guideline

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 Geo Mean 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% atthe 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) (Figure 1). 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).

Acceptability criteria:
a) At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
b) When DMSO is used as a vehicle, 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.
c) The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
d) At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
e) 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.
f) The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
g) Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

Results are unreliable when the cell count is below 5000 at non-cytotoxic concentration. If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test was rejected and repeated.
Positive control results:
Experiment 1: The positive control (TNBS) showed a S.I. ≥ 188% in all wells and was non-cytotoxic.
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 503% in all wells and was non-cytotoxic.
Experiment 3: The positive control (TNBS) showed a S.I. ≥ 162% in two out of the three wells was non-cytotoxic.
Experiment 4: The positive control (TNBS) showed a S.I. ≥ 461% in all wells and was non-cytotoxic.
Key result
Parameter:
other: EC150 (μg/mL)
Run / experiment:
Experiment 1
Value:
12
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
SI: < 88%
Positive controls validity:
valid
Key result
Parameter:
other: EC150 (μg/mL)
Run / experiment:
Experiment 2
Value:
13
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
SI: < 107%
Positive controls validity:
valid
Key result
Parameter:
other: EC150 (μg/mL)
Run / experiment:
Experiment 3
Value:
28
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
SI: < 86%
Positive controls validity:
valid
Key result
Parameter:
other: EC150 (μg/mL)
Run / experiment:
Experiment 4
Value:
20
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
SI: < 113%
Positive controls validity:
valid
Other effects / acceptance of results:
All tests 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% (99% in experiment 1, 2 and 4 and 100% in experiment 3).
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in experiment 1 and 3 and 99% in experiment 2 and 4).
- 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 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 both experiments.
- No relevant drift in CD86 expression was observed in the untreated controls (RPMI) and no drift was observed in the negative (LA) controls.

In the first experiment an induction above 150% was only observed at the highest non-cytotoxic concentration therefore led to an individual run conclusion of inconclusive.
Since the results of experiment 1 and 2 were not concordant and had the same test concentrations an additional third and fourth experiment were performed.

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

CYTOXICITY
The test item showed toxicity in all experiments.

Experiment 1
CV70: 39 μg/mL

Experiment 2
CV70: 15 μg/mL

Experiment 3
CV70: 36 μg/mL

Experiment 4
CV70: 28 μg/mL

COLOR- INTERFERENCE
(There is colour interference in the IgG1 evaluation when the X Median of the FITC fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%)

Experiment 1
Color interference was observed at 20, 50 and 100 μg/mL.

Experiment 2
Color interference was observed at 20, 50, 100 and 200 μg/mL.

Experiment 3
Color interference was observed at 30 and 60 μg/mL.

Experiment 4
Color interference was observed at 35, 40 and 50 μg/mL.
Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
Phosphoric acid, isotridecyl ester (Hostacor ITD) is classified as positive in the USENS assay since an increase (> 150%) in the expression levels of CD86 cell surface marker in the U937 cell line was oberserved under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

For skin sensitisation, the "2-out-of-3" prediction model (PM) according to Bauch et al. (2012), is the most commonly used and accepted PM to integrate the outcome of individual DPRA, KeratinoSens and U-SENS assays for a prediction of the in vivo effect of respective test substances. Using this PM, a substance is assigned as skin sensitiser if the results obtained in at least 2 of the above 3 assays are positive. In case of the discussed test item the DPRA and KeratinoSens showd negative response. Applying the "2-out-of-3" PM the discussed test item can therefore be regarded as non-sensitiser to skin also under in vivo conditions. Based here upon, the discussed test item is not subject for labelling and classification requirements regarding this endpoint.