[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

Endpoint summary

Description of key information

Studies conducted to recognised testing guidelines and GLP.

DEREK

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

KeratinoSens; OECD 442D

The objective of this study was to evaluate the ability of CMTX 2-carboxymethyloxy-thioxanthone to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD 442D guideline.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder.  The test item was dissolved in dimethyl sulfoxide at 50 mM in experiment 1 and 2 and 75 mM in experiment 3.  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 0.24 – 500 µM in experiment 1 and 2 (2-fold dilution series) and 64 – 750 µM (1.25-fold dilution series) in experiment 3.  The highest test concentration was considered to be the limit of solubility, since slightly higher concentration (1000 and 2000 µM) showed moderate precipitate in the solubility test.  No precipitate was observed at any dose level tested.  Three independent experiments were performed.

All experiments 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 (20 µM, 48 µM and 89 µM in experiment 1, 2 and 3, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (3.836-fold, 2.810-fold and 2.104-fold in experiment 1, 2 and 3, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.4%, 9.3% and 10% in experiment 1, 2 and 3, respectively).

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

In the first experiment the test item showed toxicity (IC30 value of 281 µM) 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 1.169-fold. This led to an individual run conclusion of negative. In the second experiment no clear toxicity was observed (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 1.496-fold. Since no induction or clear toxicity was observed this led to an individual run conclusion of inconclusive. As experiment 1 and 2 were not concordant an additional third experiment was performed. After a second solubility experiment a higher top test concentration of 750 µM was selected to increase the potential of finding an IC30 value in the third experiment. In the third experiment the test item showed toxicity (IC30 value of 526 µM and IC50 value of 605 µM) 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 1.393-fold. This led to an individual run conclusion of negative. Overall the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to and over the cytoxicity concentration in 2 out of 3 experiments.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

U-SENS™ Assay; OECD 442E

The objective of this study is to evaluate the ability of CMTX 2-carboxymethyloxy- thioxanthone to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The study procedures described in this report were based on the most recent OECD guideline.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder. CMTX 2-carboxymethyloxy-thioxanthone was dissolved in dimethyl sulfoxide at 50 mg/mL. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed.

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% (99% in experiment 1 and 2).

The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in experiment 1 and 2).

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 both experiments.

The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both 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 drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

CMTX 2-carboxymethyloxy-thioxanthone showed toxicity (CV70 values of 197 µg/mL and 136 µg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 8.9 µg/mL and 19 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. CMTX 2-carboxymethyloxy-thioxanthone is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

QSAR prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK NEXUS v6.0.1

2. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES: C1=CC=CC2=C1C(C=3C(S2)=CC=C(C=3)OCC(=O)O)=O

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF & QPRF below.

4. APPLICABILITY DOMAIN
See QMRF & QPRF below.

5. ADEQUACY OF THE RESULT
See QMRF & QPRF below.

Qualifier:

no guideline required

Principles of method if other than guideline:

Derek Nexus v6.0 contains 90 alerts for skin sensitisation, together with reasoning rules encoding physicochemical descriptors. In addition to a prediction of skin sensitisation potency for alerting query compounds, Derek evaluates potentially misclassified and unclassified features in compounds that do not activate skin sensitisation alerts or examples.

GLP compliance:

no

Remarks:

Full records of the QSAR model and input data are recorded in the QMRF and QPRF.

Justification for non-LLNA method:

QSAR data used as part of an in vitro testing strategy.

Specific details on test material used for the study:

Not applicable to QSAR

Parameter:

other: Structural alerts

Value:

0

Parameter:

other: Unclassified features

Value:

0

Parameter:

other: misclassified features

Value:

0

Other effects / acceptance of results:

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. CMTX (2-carboxymethyloxy-thioxanthone) is predicted to be not sensitizing to the skin.

Interpretation of results:

GHS criteria not met

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

Executive summary:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 Oct 2018 to 30 Nov 2018

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 Test Method)

Version / remarks:

Adopted June, 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

Adopted March, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%, assumed 100% for testing
Test item storage: At room temperature protected from light desiccated

Details on the study design:

Test System:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Cell Culture:
- Basic medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

- Maintenance medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum and geneticin (500 µg/mL).

- Exposure medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100% (actual range 82 to 101 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 to 37.0 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing:
Cells were subcultured upon reaching 80 to 90 % confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design:
Plating of Cells:
For testing, cells were 80 to 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+13 in experiment 1, P+3 in experiment 2 and P+7 in experiment 3.

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 items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ± 1.0 °C in the presence of 5 % CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 3 experiments were performed.

Luciferase Activity Measurement:
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 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. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate 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; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 to 4 hours at 37 ± 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.

Positive control results:

- Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.836 and the EC1.5 was 20 µM.

- Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.810 and the EC1.5 was 48 µM.

- Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.104 and the EC1.5 was 89 µM.

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.169

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.496

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: Imax

Value:

1.393

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

In the first experiment the test item showed toxicity (IC₃₀ values of 281 µM) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.169-fold. This led to an individual run conclusion of negative. In the second experiment no clear toxicity was observed (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (I_max) was 1.496-fold. Since no induction or clear toxicity was observed this led to an individual run conclusion of inconclusive. As experiment 1 and 2 were not concordant an additional third experiment was performed. After a second solubility experiment a higher top test concentration of 750 µM was selected to increase the potential of finding an IC₃₀ value in the third experiment. In the third experiment the test item showed toxicity (IC₃₀ value of 526 µM and IC₅₀ value of 605 µM) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (I_max) was 1.393-fold. This led to an individual run conclusion of negative. Overall the test item is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to and over the cytoxicity concentration in 2 out of 3 experiments.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of CMTX 2-carboxymethyloxy-thioxanthone to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens_TM assay.

The study procedures described in this report were based on the most recent OECD 442D guideline.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder.  The test item was dissolved in dimethyl sulfoxide at 50 mM in experiment 1 and 2 and 75 mM in experiment 3.  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 0.24 – 500 µM in experiment 1 and 2 (2-fold dilution series) and 64 – 750 µM (1.25-fold dilution series) in experiment 3.  The highest test concentration was considered to be the limit of solubility, since slightly higher concentration (1000 and 2000 µM) showed moderate precipitate in the solubility test.  No precipitate was observed at any dose level tested.  Three independent experiments were performed.

All experiments 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 EC_1.5 of the positive control was within two standard deviations of the historical mean (20 µM, 48 µM and 89 µM in experiment 1, 2 and 3, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (3.836-fold, 2.810-fold and 2.104-fold in experiment 1, 2 and 3, respectively).

•       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.4%, 9.3% and 10% in experiment 1, 2 and 3, respectively).

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

In the first experiment the test item showed toxicity (IC₃₀ value of 281 µM) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.169-fold. This led to an individual run conclusion of negative. In the second experiment no clear toxicity was observed (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.496-fold. Since no induction or clear toxicity was observed this led to an individual run conclusion of inconclusive. As experiment 1 and 2 were not concordant an additional third experiment was performed. After a second solubility experiment a higher top test concentration of 750 µM was selected to increase the potential of finding an IC₃₀ value in the third experiment. In the third experiment the test item showed toxicity (IC₃₀ value of 526 µM and IC₅₀ value of 605 µM) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.393-fold. This led to an individual run conclusion of negative. Overall the test item is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to and over the cytoxicity concentration in 2 out of 3 experiments.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 Feb 2019 to 01 Mar 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Annex II "In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U- SENS™)"

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 183: U937 Cell Line Activation Test for Skin Sensitization (U-SENS™)

Version / remarks:

Adopted October, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 442E; Evaluation of in vitro Skin Sensitization Potential (of the test item) with the U937 Cell Line Activation Test (U-SENS™) Assay

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Evaluation of in vitro Skin Sensitization Potential of the test item with the U937 Cell Line Activation Test (U-SENS™) Assay

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Details on the study design:

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

Stock cultures of these cells are stored in the freezer (-150°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.

Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitization to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the U-SENSTM assay, which is recommended in international guideline (e.g. OECD).

Cell Culture:
Cell culture medium:
Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

Environmental conditions:
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 21 – 101%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.4 – 38.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Other deviations in the temperature and humidity are evaluated in the raw data. Since the solvent and positive controls fulfilled the acceptability criteria, deviations in the temperature and humidity had no effect on the study integrity.

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

Number of experiments:
Two experiments were conducted per test item to demonstrate reproducibility of the results and conclusion.

Treatment of Cells:
Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.

In the second experiment cells were treated with eight selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 1.0, 2.0, 4.0, 7.5, 10, 50, 100 and 200 µg/mL.

In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.

Precipitate evaluation:
After 45 ± 3 hours of exposure, wells were checked for precipitate.

4.7.5. Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.

FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)

The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

Flow cytometry method:
Acquisition:
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.

Analysis:
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, 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 were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.

Color Interferences:
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%).

Positive control results:

See Appendix 3 attached below.

Key result

Run / experiment:

other: Test item Experiment 1

Parameter:

other: EC150

Remarks:

µg/mL

Value:

8.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Test item Experiment 1

Parameter:

other: CV70

Remarks:

µg/mL

Value:

197

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Test item Experiment 2

Parameter:

other: EC150

Remarks:

µg/mL

Value:

19

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Test item Experiment 2

Parameter:

other: CV70

Remarks:

µg/mL

Value:

136

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

The objective of this study is to evaluate the ability of CMTX 2-carboxymethyloxy-thioxanthone to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

CMTX 2-carboxymethyloxy-thioxanthone was evaluated for the ability to increase the expression levels of CD86 cell surface marker. An overview of the viability and CD86 cell surface marker activity is summarized in Table 1. The results of the positive, negative and vehicle controls are summarized in Table 2. An overview of EC₁₅₀ and CV₇₀ values is given in Table 3. The individual raw data are presented in Appendix 2.

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (98% in experiment 1 and 2, respectively). The cells were in these experiments incubated with CMTX 2-carboxymethyloxy-thioxanthone 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.

Experiment 1

• No precipitation was observed at the end of the incubation period in the 96-well plates.
• CMTX 2-carboxymethyloxy-thioxanthone showed toxicity, the calculated CV₇₀ was 197 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC₁₅₀ is 8.9 µg/mL.
• The test item showed colour interference at 20, 50, 100 and 200 µg/mL.
• The positive control (TNBS) showed a S.I. ≥ 673% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 133% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2

• No precipitation was observed at the end of the incubation period in the 96-well plates.
• CMTX 2-carboxymethyloxy-thioxanthone showed toxicity, the calculated CV70 was 136 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 19 µg/mL.
• The test item showed colour interference at 50, 100 and 200 µg/mL.
• The positive control (TNBS) showed a S.I. ≥ 583% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 122% in two out of the three wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Both 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 and 2).
• The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in experiment 1 and 2).
• 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 both experiments
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both 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 drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls. In both experiments the positive and negative control were considered valid and the positive control was within or above the historical control data (Appendix 3). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Executive summary:

The objective of this study is to evaluate the ability of CMTX 2-carboxymethyloxy- thioxanthone to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The study procedures described in this report were based on the most recent OECD 442E guideline.

The CMTX 2-carboxymethyloxy-thioxanthone was a yellow powder. CMTX 2-carboxymethyloxy-thioxanthone was dissolved in dimethyl sulfoxide at 50 mg/mL. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. Two independent experiments were performed.

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% (99% in experiment 1 and 2).
• The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in experiment 1 and 2).
• 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 both experiments.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both 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 drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

CMTX 2-carboxymethyloxy-thioxanthone showed toxicity (CV70 values of 197 µg/mL and 136 µg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 8.9 µg/mL and 19 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. CMTX 2-carboxymethyloxy-thioxanthone is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, CMTX 2-carboxymethyloxy-thioxanthone is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2019 to 18 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No 640/2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected. Weight at the Initiation of Dosing: 18.6 to 25.0 g.

Justification for Test System and Number of Animals:
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.

The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in Appendix 3 of this report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification:
At study assignment, each animal was identified using a tail mark with indelible ink.

Environmental Acclimation:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals:
Animals were assigned to the study at the discretion of the coordinating bio technician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.

Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

The disposition of all animals was documented in the study records.

Husbandry:
Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 42 to 57%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.

It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.

It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment:
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care:
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Vehicle:

dimethylformamide

Concentration:

10, 25, and 50 (test item; % w/w)

No. of animals per dose:

5

Details on study design:

Justification of Route and Dose Levels:
Dose route and dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.

Pre-screen Test:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic
toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that
can technically be applied.

Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study
except that the assessment of lymph node proliferation and necropsy were not performed.

Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolo cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1and 3, and on Day 6.

Animals were sacrificed after the final observation.

Main Study:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Three groups of five animals were treated with one test item concentration per group. One group of five animals was treated with vehicle.

Allocation:

Text table 1
Allocation
Group[1] animal numbers induction (test item; % w/w)
1 01 - 05 Vehicle control 0 (N,N-dimethylformamide (DMF))
2 06 - 10 Experimental low concentration 10
3 11 - 15 Experimental Intermediate concentration 25
4 16 - 20 Experimental high concentration 50
[1] five females per group

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized by intraperitoneal injection (0.2mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6:
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In-life Procedures, Observations, and Measurements:
Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations:
Postdose Observations:
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Body Weights:
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation:
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Erythema and eschar formation:
No erythema .............................................................................................................................................................0
Very slight erythema (barely perceptible) ...............................................................................................................1
Well-defined erythema .............................................................................................................................................2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) ..................................3
Severe erythema (beet redness) to eschar formation preventing grading of erythema ........................................4

Terminal procedures:
No necropsy was performed, since all animals survived until the end of the observation period.

Statistics:

See below

Positive control results:

Not applicable

Key result

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

Group 1 0% Test substance w/w

Key result

Parameter:

SI

Value:

1.5

Variability:

± 0.2

Test group / Remarks:

Group 2 10% Test substance w/w

Key result

Parameter:

SI

Value:

2.3

Variability:

± 0.5

Test group / Remarks:

Group 3 25% Test substance w/w

Key result

Parameter:

SI

Value:

1.1

Variability:

± 0.2

Test group / Remarks:

Group 4 50% Test substance w/w

For detailed results see Appendix 1.

Pre-screen Test

At a 25 and 50% test item concentration, no signs of systemic toxicity were noted and up to very slight irritation was observed. Therefore, a 50% concentration was selected as highest concentration for the main study.

Main Study

Skin Reactions / Irritation

The very slight erythema of the ears as shown by animals treated at 25 and 50% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes.

Yellow test item remnants were present on the dorsal surface of the ears of test item treated animals between Days 1 and 5, which did not hamper scoring of the skin reactions.

Systemic Toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 867, 1269 and 635 DPM, respectively. The mean DPM/animal value for the vehicle control group was 563 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.5, 2.3 and 1.1, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 50%, CMTX 2-carboxymethyloxy-thioxanthone was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%.

The response of the 50% group did not follow the expected dose-response relationship which more often seen in these kind of studies. The response might be less due to differences in skin penetration or viscosity.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).

Based on these results, CMTX 2-carboxymethyloxy-thioxanthone would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether CMTX 2-carboxymethyloxy-thioxanthone induces skin sensitization in CBA/J mice after three epidermal exposures of the animals under the conditions described in this report.

The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010).

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 50% test item concentration, no signs of systemic toxicity were noted and up to very slight irritation was observed. Therefore, a 50% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide (DMF)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 867, 1269 and 635 DPM, respectively. The mean DPM/animal value for the vehicle control group was 563 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.5, 2.3 and 1.1, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 50%, CMTX 2-carboxymethyloxy-thioxanthone was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%. The response of the 50% group did not follow the expected dose-response relationship which more often seen in these kind of studies. The response might be less due to differences in skin penetration or viscosity.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, CMTX 2-carboxymethyloxy-thioxanthone would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)