Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

In a study conducted according to OECD 442D and in compliance with GLP, using the ARE-Nrf2 luciferase KeratinoSens™ test method, di-tert-nonyl polysulfides induced luciferase activation in three out of four experiments (EC₁.₅ values of 29, 104 and 6.8, Imax values 2.84, 1.87, and 2.76 in experiments 1, 2 and 4). In two of the four experiments (1 and 4) the results were concordant: the responses were dose-related and no toxicity was observed; in experiment 2 the response was biphasic and no toxicity was observed so it was concluded that the test substance is positive under the conditions of the study (Charles River Laboratories, 2021a).

In a study conducted according to OECD 442E and in compliance with GLP, using the U-SENS™ method which quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, di-tert-nonyl polysulfides induced CD86 activity in both experiments (EC150 values of 12 μg/mL and 26 μg/mL in experiment 1 and 2, respectively), and is therefore classified as positive in this assay (Charles River Laboratories, 2021b).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Dec 2020 to 21 Jan 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
ARE-Nrf2 luciferase KeratinoSens™ test method
Details of test system:
Keratinoses transgenic cell line [442D]
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: the test item was dissolved in ethanol at 40 mg/mL
- Preparation of the test chemical serial dilutions from the stock solution: 11 spike solutions in ethanol were prepared (2-fold in the first experiment and 1.5-fold dilution series in the second, third and fourth experiment). The stock and spike solutions were diluted 100-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay to give the final test concentrations.
- Preparation of the positive controls: The positive control used in the case of KeratinoSensTM is ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.
- Preparation of the solvent, vehicle and negative controls: The vehicle control was 0.25% ethanol and the negative control was 1% DMSO, both in exposure medium. Eighteen wells were tested per plate per vehicle control. On each plate three blank wells were tested (no cells and no treatment).
- Stable dispersion obtained: The test item precipitated at dose levels of 25, 8.8, 13 and 13 μg/mL and upwards in experiment 1, 2, 3 and 4, respectively at the start and end of the incubation period in the 96-well plates.

DOSE RANGE FINDING ASSAY:
- Highest concentration used : 100 µg/ml
- Solubility in solvents : The test item could not be dissolved in Milli-Q water or in DMSO at a concentration of 40 mg/mL. The test item was suspended in ethanol to a final concentration of 160 mg/mL (yellow suspension which precipitated quickly into an emulsion). The test item was suspended in ethanol to a final concentration of 80 mg/mL (yellow suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
- Solubility in incubation medium : The 100-fold dilution in DMEM glutamax of 160 and 80 mg/mL showed moderate precipitate and were therefore not suitable to test. The 100-fold dilution of the 40 mg/mL ethanol stock showed slight precipitation. This concentration was selected as highest concentration for the main assay (limit of solubility).
- Cytotoxicity assessment performed : no
- Final concentration range selected on basis of: limit of solubility

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates : 3
- Number of repetitions: 4
- Test chemical concentrations : of 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.2, 0.1 and 0.05 μg/mL (final concentration ethanol of 0.25%) in the first experiment and 100, 67, 44, 30, 20, 13, 8.8, 5.9, 3.9, 2.6, 1.7 and 1.2 μg/mL (final concentration ethanol of 0.25%) in the second, third and fourth experiment. Test item concentrations were used within 3 hours after preparation.
- Application procedure : Four experiments, duplicate plates, 25-fold diluted test chemical and control items. Three wells per plate were left empty (no cells and no treatment) to assess background values. Initially, experiment 1 was terminated due to infections. In addition, experiment 3 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 4 valid experiments were performed.
- Exposure time : 48 hours ± 1 hour
- Study evaluation and decision criteria used :
The prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is equal or higher than (≥) 1.5 fold and statistically significantly different as compared to the vehicle (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 ≥ 1.5 fold (i.e. at the EC₁.₅ determining concentration);
3. The Imax 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 and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.
- Description on study acceptance criteria :
• The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
• The EC₁.₅ of the positive control should be within two standard deviations of the historical mean. 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 vehicle control DMSO, and, if applicable, Milli-Q or ethanol, should be below 20% in each repetition which consists of 18 wells. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density) : passage number: P+5, P+7, P+8 and P+10 in experiment 1, 2, 3 and 4, respectively. Seeding density: 10,000 cells/well in duplicate 96-well plates. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay.
- Incubation conditions : 37±1.0°C in the presence of 5% CO²
- Precipitation noted : at start and end of exposure period at 25 μg/mL (Expt. 1), 8.8 μg/mL (Expt 2) and 13 μg/mL (Expt 3 and 4)

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test : TECAN Infinite® M200 Pro Plate Reader (integration time 2 minutes)
- Plate used :96-well plates
- Lysate preparation : The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL (100 in Experiment 2) 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 5 minutes at room temperature. The lower volume was considered to have no effect on the outcome as the reagent is added only for luminescence readings and the solvent and positive controls fulfill the criteria.

DATA EVALUATION
- Cytotoxicity assessment : medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
- Prediction model used : See evaluation criteria above and prediction model Figure attached.


Vehicle / solvent control:
other: ethanol and DMSO
Negative control:
other: DMSO
Positive control:
EGDMA (120 M) [442D]
Positive control results:
A dose-related induction of luciferase activity was observed in all experiments:
Experiment 1: Iₘₐₓ 2.45, EC₁.₅ 46 µM
Experiment 2: Iₘₐₓ 2.66, EC₁.₅ 104 µM
Experiment 3: Iₘₐₓ 3.72, EC₁.₅ 35 µM
Experiment 4: Iₘₐₓ 3.84, EC₁.₅ 49 µM
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Value:
29 µM
Cell viability:
The test item showed no toxicity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Dose-related induction of luciferase activity
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
2.84 %
Cell viability:
The test item showed no toxicity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Dose-related induction of luciferase activity
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Value:
1.1 µM
Cell viability:
Toxicity was observed
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
response was biphasic and non dose related response
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
1.87 %
Cell viability:
Toxicity was observed
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
response was biphasic and non dose related response
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC30 [442D]
Value:
47 mM
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC50 [442D]
Value:
86 mM
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
1.38 %
Cell viability:
The test item showed no toxicity.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
other: run/experiment 4
Parameter:
EC 1.5 [442D]
Value:
6.8 µg/mL
Cell viability:
The test item showed no toxicity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Dose-related induction of luciferase activity
Key result
Group:
test chemical
Run / experiment:
other: run/experiment 4
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
2.76 %
Cell viability:
No test item toxicity was observed
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Dose-related induction of luciferase activity
Group:
other: positive control
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Value:
46 µM
Group:
other: positive control
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
2.45 %
Group:
other: positive control
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Value:
1.4 µM
Group:
other: positive control
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
2.66 %
Group:
other: positive control
Run / experiment:
run/experiment 3
Parameter:
EC 1.5 [442D]
Value:
35 µM
Group:
other: positive control
Run / experiment:
run/experiment 3
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
3.72 %
Group:
other: positive control
Run / experiment:
other: run/experiment 4
Parameter:
EC 1.5 [442D]
Value:
49 µM
Group:
other: positive control
Run / experiment:
other: run/experiment 4
Parameter:
Imax [442D]
Remarks:
Imax is unitless
Value:
3.84 %
Outcome of the prediction model:
positive [in vitro/in chemico]
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: historical control data are presented in summary, and duplicate vehicle control data are given in the report but data for reference controls are not given.
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Experiment 1

•Precipitation was observed at the start and end of the incubation period in the 96-well plates at concentrations of 25 μg/mL and upwards.

•The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated.

•A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.84 and the EC₁.₅ 29 μg/mL.

•The positive control ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.45 and the EC₁.₅ 46 μM.

Experiment 2

•Precipitation was observed at the start and end of the incubation period in the 96-well plates at concentrations of 8.8 μg/mL and upwards.

•The test item showed toxicity. The calculated IC₃₀ was 47 μg/mL and the calculated IC₅₀ was 86 μg/mL.

•An induction was observed after treatment with the test item. The Imax was 1.87 and the EC₁.₅1.1 μg/mL.

•The positive control ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.66 and the EC₁.₅ 104 μM.

Experiment 3

•Precipitation was observed at the start and end of the incubation period in the 96-well

plates at concentrations of 13 μg/mL and upwards.

•The test item showed no toxicity. The viability of the cells was higher than 70% at all test

concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated.

•No luminescence activity induction compared to the vehicle control was observed at any

of the test concentrations after treatment with the test item. The Imax was 1.38 and

therefore no EC₁.₅ could be calculated.

•The positive control ethylene dimethacrylate glycol caused a dose related induction of

the luciferase activity. The Imax was 3.72 and the EC₁.₅ 35 μM.

Experiment 4

•Precipitation was observed at the start and end of the incubation period in the 96-well

plates at concentrations of 13 μg/mL and upwards.

•The test item showed no toxicity. The viability of the cells was higher than 70% at all test

concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated.

•A dose related luminescence activity induction was observed after treatment with the test

item. The Imax was 2.76 and the EC₁.₅ 6.8 μg/mL.

•The positive control ethylene dimethacrylate glycol caused a dose related induction of

the luciferase activity. The Imax was 3.84 and the EC₁.₅ 49 μM.

All tests passed the acceptance criteria:

•The luciferase activity induction obtained with the positive control, Ethylene

dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at

least one concentration.

•The EC₁.₅ of the positive control was within two standard deviations of the historical

mean (46 μM, 104 μM, 35 μM and 49 μM in experiment 1, 2, 3 and 4, respectively). A

dose response was observed and the induction at 250 μM was higher than 2-fold (2.45-

fold, 2.66-fold, 3.72-fold and 3.84-fold in experiment 1, 2, 3 and 4, respectively).

•The average coefficient of variation of the luminescence reading for the vehicle

(negative) control ethanol was below 20% (8.5%, 9.2%, 6.6% and 10% in experiment 1,

2, 3 and 4, respectively).

•Finally, the average coefficient of variation of the luminescence reading for the vehicle

(negative) control DMSO was below 20% (6.9%, 14%, 5.4% and 13% in experiment 1,

2, 3 and 4, respectively).

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In a study conducted according to OECD 442D and in compliance with GLP using the ARE-Nrf2 luciferase KeratinoSens™ test method, di-tert-nonyl polysulfides induced luciferase activation in three out of four experiments (EC₁.₅ values of 29, 104 and 6.8, Imax values 2.84, 1.87, and 2.76 in experiments 1, 2 and 4). In two of the four experiments (1 and 4) the results were concordant: the responses were dose-related, and no toxicity was observed, in experiment 2 the response was biphasic and no toxicity was observed so it was concluded that the test substance is positive under the conditions of the study.
Executive summary:

The ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was examined with the aid of luciferase in the KeratinoSens™ cell line in an assay conducted according to OECD 442D and in compliance with GLP.

The test item was dissolved in ethanol at 40 mg/mL. From this stock 11 spike solutions in ethanol were prepared. The stock and spike solutions were diluted 400-fold in the assay resulting in test concentrations of 0.05 – 100 μg/mL (2-fold dilution series) in the first experiment and resulting in test concentrations of 1.2 – 100 μg/mL (1.5-fold dilution series) in the second, third and fourth experiment. A more narrow dose-response analysis was performed using a lower dilution factor of 1.5-fold to investigate the induction at 100 μM in experiment 1 in more detail. The highest test concentration was considered to be the limit of solubility. The test item precipitated at concentrations of 25, 8.8, 13 and 13 μg/mL and upwards in experiment 1, 2, 3 and 4, respectively. Four independent experiments were performed.

In the first experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 29 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.84-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control.

In the second experiment, the test item showed toxicity (IC30 value of 47 μg/mL and IC50 value of 86 μg/mL). An induction of the luciferase activity (EC1.5 value of 1.1 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 1.87-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control. However, a biphasic non dose related response was observed.

Since the first two experiments were not concordant, a third experiment was performed to provide a final conclusion.

In the third experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.38-fold leading to an individual run conclusion of inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control.

Since the third experiment does not give a decisive answer, a fourth experiment was performed to provide a final conclusion.

In the fourth experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 6.8 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.76-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control.

Overall, positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control in three out of four experiments (one individual run was biphasic but positive nonetheless).

It was concluded that di-tert-nonyl polysulfides (TNPS 537) is positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 May 2021 to 21 May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
U937 cell line activation test (U-SENS™)
Details of test system:
U-937 cell line [442E]
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: the test item was dissolved in 0.2% ethanol at 100 mg/mL
- Preparation of the test chemical serial dilutions: 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 ethanol of 0.2%). Test item concentrations were used within 2 hours after preparation.
- Preparation of the positive controls: a 10 mg/mL solution was prepared in complete medium (RPMI). This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 μg/mL).
- Preparation of the solvent, vehicle and negative controls: No details are given of solvent or vehicle control preparation. A lactic acid (negative control) solution at 10 mg/mL was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 μg/mL).
- Stable dispersion obtained: no information on stability in solvent
- Log Kow of the test chemical: not given in report.

SOLUBILITY ASSAY :
- Highest concentration used: 50 mg/mL in RPMI and in DMSO. 100 mg/mL in ethanol
- Solubility in solvents: an emulsion was formed in RPMI and in DMSO. A clear yellow solution was formed in ethanol.
- Solubility in incubation medium: no precipitation was observed at the end of the incubation period in the 96-well plates.

- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: CV70

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: two (test item, one for non-specific IgG1 binding, the other for CD86 binding); three replicates of untreated control (RPMI),
vehicle control (EtOH), negative (LA) and positive (TNBS) controls
- Number of repetitions: The experiment was performed twice.
- Test chemical concentrations: 1.0, 10, 20, 50, 100 and 200 μg/mL (first and second experiment).
- Application procedure: cells were treated with 100 µL of selected doses or controls. Final volume 200 µL in 96-well plates
- Exposure time: 45 ± 3 hours
- Study evaluation and decision criteria used: cytotoxicity evaluated by CV70, parameters set on the RPMI wells for IgG1 and used for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity was analyzed as percentage of cells in P2. Stimulation index (S.I.) was evaluated by plotting the P2 region in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant is placed. The percentage of cells in the UR quadrant will be used to calculate the stimulation index.
- Description on study acceptance 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 EtOH vehicle control is assessed by calculating a EtOH S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The EtOH 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%. If one IgG1 level of a complete medium control is outside the range, it is discarded.
- 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 as valid if at least two out of the three wells are positive (CD86-IgG1 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
- 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%).
In addition, for each vehicle control (complete medium and/or DMSO), the CD86-IgG1 % positive cells of the three wells is averaged. If one control is > 25% above or below the mean, it is an outlier to be discarded (if more than one outlier is identified, only the one furthest away from the mean is discarded). If one complete medium and/or DMSO control outlier is discarded, the corrected CD86-IgG1 % of the two remaining complete medium and/or DMSO controls should be ≤ 25% above or below their mean.


SEEDING AND INCUBATION
- Seeding conditions: (passage number and seeding density): 5.0 x 10⁵ viable cells/mL exposed to test material.
- Incubation conditions: humid atmosphere, 80 - 100%, containing 5.0 ± 0.5% CO₂ in air in the dark at 37.0 ± 1.0°C.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- Antibodies used: Mouse IgG1 of unknown specificity for isotypic control; Human CD86 specific mouse IgG1 for measurement of CD86 expression.
- IgG1 and CD86 staining: cells were transferred into a new 96-well plate containing 5 µl of antibody (1:1 diluted in PBS) and refrigerated in the dark for at least 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.
- Flow cytometry used: yes, BD FACSCanto™ flow cytometer.
- Plate used: V-shaped 96-well plates
- Propidium iodide staining/cytotoxicity measurements: Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution was added to each well. The size (FSC) is set linear and the granularity (SSC) parameter is set to logarithmic scale and a R1 region was defined in which approximately 10,000 events are acquired for each culture. The acquisition parameters will remain unchanged for the acquisition of all the wells.
- Preparation for CD54 and/or CD86 expression measurements/cell staining: Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g, 4°C). The supernatant was discarded, and cells rinsed once with Phosphate Buffered Saline (PBS) containing 5% FCS. After washing there was a second centrifugation step, and 100 μL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.


DATA EVALUATION
- Cytotoxicity assessment:
- Colour 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%).
- Prediction model used:
- 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 cytotoxic concentration and the lowest cytotoxic 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).
Vehicle / solvent control:
other: 0.2% ethanol in complete medium
Negative control:
DL-Lactic acid
Positive control:
other: 2,4,6-trinitrobenzenesulfonic acid
Positive control results:
Experiment 1: The positive control (TNBS) showed a S.I. of ≥ 452 in all wells and was non-cytotoxic (cell viability ≥ 70%), when tested at a concentration of 50 µg/mL
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 737% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%), when tested at a concentration of 50 µg/mL.
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC150, CD86 [442E]
Value:
12 %
Cell viability:
No cytotoxicity observed
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC150, CD86 [442E]
Value:
26 %
Cell viability:
No cytotoxicy observed
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
87 %
At concentration:
1 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
131 %
At concentration:
10 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
240 %
At concentration:
20 other: µg/mL
Cell viability:
100 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
104 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
185 %
At concentration:
100 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
333 %
At concentration:
200 other: µg/mL
Cell viability:
99%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
100 %
At concentration:
1 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
94 %
At concentration:
10 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
135 %
At concentration:
20 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
206 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
400 %
At concentration:
100 other: µg/mL
Cell viability:
99%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
706 %
At concentration:
200 other: µg/mL
Cell viability:
99%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 1
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
452 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 2
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
632 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 3
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
619 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 1
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
743 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 2
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
737 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
other: positive control TNBS 3
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
789 %
At concentration:
50 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Group:
other: negative control 1
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
90 %
At concentration:
200 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: negative control 2
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
65 %
At concentration:
200 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: negative control 3
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
103 %
At concentration:
200 other: negative control stimulation index µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: negative control 1
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
39 %
At concentration:
200 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: negative control 2
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
72 %
At concentration:
200 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: negative control 3
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
91 %
At concentration:
200 other: µg/mL
Cell viability:
100%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: solvent control (mean)
Run / experiment:
run/experiment 1
Parameter:
other: stimulation index
Value:
118 %
At concentration:
0.2 other: %
Cell viability:
100%
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Group:
other: solvent control (mean)
Run / experiment:
run/experiment 2
Parameter:
other: stimulation index
Value:
111 %
At concentration:
0.2 other: %
Cell viability:
100%
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Outcome of the prediction model:
positive [in vitro/in chemico]

Both 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% in experiment 1 and 2).

• The mean viability of the triplicate EtOH vehicle control cells was above the threshold of 90% (100% in experiment 1 and 2).

• The EtOH vehicle control mean value of its triplicate CD86-IgG1 S.I. was smaller than 250% of the mean of the triplicate CD86-IgG1 S.I. of untreated U937 cells in both experiments.

• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments. In the first experiment, one of the three values was below 2% (i.e. 1.5). Since RPMI2 was excluded from analysis based on CD86-IgG1 expression value > 25% from mean, this minor deviation has no effect on the study outcome.

• 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 drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive (TNBS) and negative (LA) controls were considered valid.

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity (No CV70 value). An induction of the CD86 activity (EC150 values of 12 μg/mL and 26 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. 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.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In a study conducted according to OECD 442E and in compliance with GLP using the U-SENS™ method which quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, di-tert-nonyl polysulfides induced CD86 activity in both experiments (EC150 values of 12 μg/mL and 26 μg/mL in experiment 1 and 2, respectively), and is therefore classified as positive in this assay.
Executive summary:

The ability of the test item to increase the expression levels of the CD86 cell surface marker was evaluated in the U937 cell line activation Test (U-Sens™) assay in a study conducted according to OECD 442E and in compliance with GLP. The test item was evaluated for the ability to increase the expression levels of CD86 cell surface marker.

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (94% and 99% in experiment 1 and 2, respectively). The cells were in these experiments incubated with the test item in a concentration range of 1.0 – 200 μg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

No precipitation was observed, and the test item showed no toxicity in either experiment, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 μg/mL. The test item showed colour interference in both experiments at 100 and 200 μg/mL.

The positive control (TNBS) showed an S.I. ≥ 452% (experiment 1) and an S.I. ≥ 737% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 103% (experiment 1) and an S.I. 91% (experiment 2) in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, with EC150 values of 12 μg/mL and 26 μg/mL in experiment 1 and 2, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In vivo studies

There are two available in vivo studies which evaluate the skin sensitizing potential of di-tert-nonyl polysulfides.  

In the more recent skin sensitization test (Manciaux, 2002), male and female Hartley guinea pigs (10/sex) were exposed to di-tert-nonyl polysulfides (TPS 37 LS; CAS # 68425-16-1). On day 1 of the induction phase, all animals in the treatment group were administered the following 3 pairs of intradermal injections in the intrascapular region: (1) Freund’s complete adjuvant (FCA) 50% v/v in 0.9% NaCl; (2) TPS 37 LS (25% w/w in corn oil; and (3) TPS 37 LS (25% w/w in corn oil) in a 50% v/v mixture of FCA and 0.9% NaCl. Animals in the control group were administered corn oil using a dosing regimen identical to the one described above. On day 8 of the induction phase, animals in the treatment group were topically administered TPS 37 LS (at the same site) at a concentration of 50% (w/w) in acetone while animals in the control group were topically administered the vehicle control acetone. The application sites were subsequently covered with occlusive dressing for a period of 48 hours. On day 22, all animals of the test and control group were challenged by a cutaneous application of TPS 37 LS (1% w/w in acetone) to the right flank. The left flank served as control and received the vehicle only. Subsequently, the test material and vehicle control were maintained under an occlusive dressing for a period of 24 hours and skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. No mortality or signs of adverse clinical toxicity were observed at challenge or through the study period. No positive reactions were noted for any of the test (0/20) or control animals (0/10) at 24 and 48 hours. Based on these results, TPS 37 LS was not found to be sensitizing in the guinea pig maximization test.

In an older study (Gonnet, 1979), male and female Hartley guinea pigs (10/sex) were exposed to di-tert-nonyl polysulfides. In the induction phase, the test substance was administered topically at 50% in olive oil 10 times, 3 days per week at 2-day intervals and Freund's complete adjuvant was administered intradermally on days 1 and 10. The test sites were covered with an occlusive patch until day 24, 48 hours after the last administration. On day 36, all animals were challenged by a cutaneous application of 50% test substance in olive oil. The test sites were covered with an occlusive patch for 48 hours. Skin reactions were observed in 9 out of 20 animals at 1, 6, 24 and 48 hours after patch removal. Histopathological evidence of an allergic reaction was found in 9 out of 10 skin samples taken from these animals for histological evaluation. No data on positive or vehicle control were included.

In vitro data

The available in vivo skin sensitization studies give conflicting results, and the more recent of these (Manciaux, 2002) is considered to be inadequate as the maximum concentration tested at challenge was 1%, therefore in vitro studies were conducted.

The ability of di-tert-nonyl polysulfides to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was examined with the aid of luciferase in the KeratinoSens™ cell line in an assay conducted according to OECD 442D and in compliance with GLP. This assay addresses Key Event 2 of the Adverse Outcome Pathway for skin sensitization (OECD, 2012).

Test concentrations: 0.05 – 100 μg/mL (experiment 1) and 1.2 – 100 μg/mL in the second, third and fourth experiment.

The highest test concentration was considered to be the limit of solubility. The test item precipitated at dose levels of 25, 8.8, 13 and 13 μg/mL and upwards in experiment 1, 2, 3 and 4, respectively. Four independent experiments were performed.

In the first experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 29 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.84-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations <200 μg/mL with a cell viability of >70% compared to the vehicle control.

In the second experiment, the test item showed toxicity (IC30 value of 47 μg/mL and IC50 value of 86 μg/mL). An induction of the luciferase activity (EC1.5 value of 1.1 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 1.87-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations <200 μg/mL with a cell viability of >70% compared to the vehicle control. However, a biphasic non dose related response was observed.

Since the first two experiments were not concordant, a third experiment was performed in an attempt to provide a final conclusion.

In the third experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.38-fold leading to an individual run conclusion of inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations at test concentrations <200 μg/mL with a cell viability of >70% compared to the vehicle control.

Since the third experiment did not give a decisive answer, a fourth experiment was performed to provide a final conclusion.

In the fourth experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 6.8 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.76-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations <200 μg/mL with a cell viability of >70% compared to the vehicle control.

Overall, positive results (>1.5-fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control in three out of four experiments (one individual run was biphasic but positive nonetheless).

It was concluded that di-tert-nonyl polysulfides (TNPS 537) is positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

The ability of di-tert-nonyl polysulfides to increase the expression levels of the CD86 cell surface marker was evaluated in the human histiocytic lymphoma U937 cell line activation Test (U-Sens™) assay in a study conducted according to OECD 442E and in compliance with GLP. This study quantifies changes of CD86 cell surface marker expression on this cell line and addresses Key Event 3 of the Adverse Outcome Pathway. The test item was evaluated for the ability to increase the expression levels of CD86 cell surface marker.

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (94% and 99% in experiment 1 and 2, respectively). The cells were in these experiments incubated with the test item in a concentration range of 1.0 – 200 μg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

No precipitation was observed, and the test item showed no toxicity in either experiment, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 μg/mL. The test item showed color interference in both experiments at 100 and 200 μg/mL.

The positive control (TNBS) showed an S.I. ≥452% (experiment 1) and an S.I. ≥737% in all wells and was non-cytotoxic at all concentrations (cell viability ≥70%). The negative control (LA) showed a S.I. ≤103% (experiment 1) and an S.I. ≥91% (experiment 2) in all wells and was non-cytotoxic at all concentrations (cell viability ≥70%).

A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, with EC150 values of 12 μg/mL and 26 μg/mL in experiment 1 and 2, respectively.

In silico prediction of sensitization

Prediction of sensitization was carried out using the OECD QSAR Toolbox (https://www.qsartoolbox.org) following the protocol shown in Appendix 2 of OECD Test Guideline 497. As the target chemical used in the prediction cannot be a UVCB, representative smiles strings for the main constituents of TNPS were run as separate targets. Provided a prediction falls within the domain, it can be taken into account in the consideration of classification for sensitization.

The constituents of TNPS fall within the parametric domain. The example structures used in the prediction were as follows:

TNPS Constituent S3       CCC(CC(SSSC(CC(C(C)C)C)(C)C)(CC)C)C              

TNPS Constituent S4       CCCCC(C(SSSSC(CC(C(C)C)C)(C)C)(C)C)C

TNPS Constituent S5       CCCCC(C(SSSSSC(CC(CC)C)(CC)C)(C)C)C

TNPS Constituent S6       CCC(CC(SSSSSSC(CC(C(C)C)C)(C)C)(CC)C)C

TNPS Constituent S9       CCCCC(C(SSSSSSSSSC(CC(C(C)C)C)(C)C)(C)C)C.

All the constituents were predicted to be negative for skin sensitization using the DASS AW in OECD QSAR Toolbox 4.4.

Consideration of Adverse Outcome Pathway (AOP)

To evaluate the skin sensitization potential of di-tert-nonyl polysulfides, the skin sensitization Adverse Outcome Pathway (AOP) was considered. The AOP focuses on chemicals that react with amino acid residues (i.e. cysteine or lysine) such as organic chemicals (OECD Test Guideline 497).

The first key event in the AOP is the covalent binding of electrophilic substances to nucleophilic centers in skin proteins. The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as changes in gene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The third key event is the activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines. The fourth key event is T-cell proliferation, and the adverse outcome is presentation of allergic contact dermatitis.

The "2 out of 3" defined approach for skin sensitization hazard identification described in OECD Test Guideline 497 (2021) consists in assessing two of the first three Key Events of the skin sensitization Adverse Outcome Pathway (AOP). Key events 2 and 3 have been evaluated for di-tert-nonyl polysulfides.

Key Event 2) Effects in keratinocytes. Di-tert-nonyl polysulfides was positive in the KeratinoSens™ assay (Charles River Laboratories, 2021a) and therefore considered to have potential to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

Key Event 3) Effects in dendritic cells. Di-tert-nonyl polysulfides was positive in the U-SENS™ assay (Charles River Laboratories, 2021b) and therefore considered to have potential to increase the expression levels of the CD86 cell surface marker.

Following the Data Interpretation Procedure described in OECD Test Guideline 497, which states that two concordant results obtained from methods addressing at least two of the first three Key Events of the AOP determine the final classification, di-tert-nonyl polysulfides is positive for skin sensitization and should be classified as a skin sensitizer, Category 1.

Consideration of potency

The result of the older in vivo study, (Gonnet, 1979), is positive: the study authors concluded that the test material produced a 45% (9/20) cutaneous skin sensitization rate in the male and female albino guinea pigs. This was a Guinea pig maximization test (GPMT) (distinguished from a Buehler test by use of an adjuvant), with topical application of the test substance in the induction and challenge phase, which does not fit the criteria shown in Tables 3.4.3 and 3.4.4 of the CLP Regulation (EC) No. 1272/2008, which assumes induction in a GPMT will be by intradermal injection (see also OECD Test Guideline 406). In the study intradermal injection was used only for the adjuvant. However, if similar concentration criteria (Cat 1B: ≥30 % responding at >1 % intradermal induction dose) were applied to topical induction, this study with 45% positive responses at an induction dose of 50% would support classification as Category 1B. In view of the deficiencies of the study, this study was not considered key, but it provides information on potency and supports a classification as Category 1B.

Integrated testing strategy (ITS) Defined Approach v1 Data Interpretation Procedure (DIP)

This approach takes the results of Key Event 1 and Key Event 3 studies and combines with in silico prediction to provide information on potency, based on the defined approaches described in OECD Test Guideline 497.

Key Event 1 could not be assessed for di-tert-nonyl polysulfides because it is a UVCB substance so in chemico studies could not be conducted, however information on Key Event 3 is available from the OECD Test Guideline 442E study. This guideline provides methods for three different tests to evaluate activation of dendritic cells: the Human Cell Line Activation test (h-CLAT), the U937 cell line activation Test (U-SENS™) and the Interleukin-8 Reporter Gene Assay (IL-8 Luc) assay. The result of this assay was positive with EC150 values of 12 μg/ml and 26 μg/ml. The DIP assigns a score based on the minimum induction threshold (MIT) which is the minimum EC150 value. The criteria presented in Table 3.1 for the h-CLAT test (p24 of OECD Test Guideline 497) have been applied to the results of the U-Sens™ assay giving a score of 2 based on the lowest EC150 value of 12 μg/ml.

Based on Figure 3.1 (p27 of OECD Test Guideline 497), this score was combined with the OECD Toolbox prediction of negative (score 0). The result was a combined score of 2 and a prediction of UN GHS 1B.

OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Organisation for Economic Cooperation and Development, Paris.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In vitro studies evaluating Key Events 2 and 3 of the Adverse Outcome Pathway for skin sensitization gave positive results, so it is concluded that di-tert-nonyl polysulfides is a skin sensitiser in vitro. Based on these results, a prediction of negative for sensitization by OECD Toolbox and the potency data available from the older in vivo study, di-tert-nonyl polysulfides meets the criteria for classification as a dermal sensitizer, Category 1B, H317 "may cause an allergic skin reaction" according to CLP Regulation (EC) No 1272/2008.