Registration Dossier

Administrative data

Description of key information

A DEREK assessment, DPRA assay and KeratinoSens assay were performed. Based on the results obtained, the substance is considered to be a skin sensitizer classified with Category 1A.

Trimethyl hexamethylene Diisocyanate, a reactant, remains present at a concentration of 8% after the substance is manufactured. Trimethyl hexamethylene Diisocyanate has a health hazard classification, respiratory sensitizer (H334), that has not been investigated because there is no requirement to do so in accordance with Annex VII of the REACH Regulation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
In silico model prediction
Type of information:
(Q)SAR
Remarks:
DEREK NEXUS model
Adequacy of study:
weight of evidence
Study period:
29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
1. SOFTWARE
: DEREK NEXUS

2. MODEL (incl. version number)
: DEREK NEXU - skin sensitization - Version 6.0.1.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
: O=C(NCCC(C)(C)CC(C)CN=C=O)SCC[Si](OC)(OC)OC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint:
OECD Principle 1. Skin sensitization. Dependent variable: data from several toxicity assays (e.g. LLNA, GPMT, HRIPT) and mechanistic studies (e.g. DPRA) are synthesised to arrive at an expert conclusion on whether compounds within the model training set are likely to be a skin sensitizer.
- Unambiguous algorithm:
OECD Principle 2. The QMRF DEREK NEXUS 6.0.1 - skin sensitization was prepared by the provider LHASA Ltd. (UK). Skin sensitization is plausible for alert 409 (isocyanate). All constituents of the substance contain an isocyanate and based on this substructure the alert is presented for constituent A. As the alert fired due to the isocyanate group, and taking into consideration that substituents on the carbons next to this group are irrelevant for the prediction, the result of constituent A can be extrapolated to the other three constituents of the thioisocyanate adduct. DEREK prediction does take stereochemistry into account, if evidence of its influence on reactivity has been presented in literature; otherwise only 2D connectivity is taken into consideration. The current substance is racemic. The stereoisomerism is not expected to affect the reliability of EC3 prediction of the substance, as the mechanism that operates within this alert is unaffected by stereoisomerism.
- Defined domain of applicability:
OECD Principle 3.
- Appropriate measures of goodness-of-fit and robustness and predictivity: OECD Principle 4.
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 76%. The alert was plausible for this substance; the weight of evidence supports the proposition
- Mechanistic interpretation:
OECD Principle 5. The substance may act as an electrophilic carbamylating agent.

5. APPLICABILITY DOMAIN
- Structural and mechanistic domains:
The substance contains an isocyanate which falls within the structure fragment for alert 409. The potential mechanism is thought to occur via carbamylation of the sulphydryl group of cysteine, consequently forming a protein-hapten complex triggering skin sensitization. No metabolization of the substance is needed for skin sensitization regarding the scope of the mechanism under which this substance is possibly skin sensitizing.
- Similarity with analogues in the training set:
up to 34%.
- Other considerations (as appropriate):
Considerations on structural analogues: all similar compounds contain one or two isocyanates substituted to either an alkyl chain or a benzene ring. All these compounds fall within the scope of the alert 409. The query compound contains an isocyanate substituted to an alkyl chain in all four constituents. All most similar compounds, are classified as a strong or extreme skin sensitizer (EC 3 < 2.0%).

6. ADEQUACY OF THE RESULT
The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments. This result can be directly used within a weight-of-evidence approach together with in chemico/in vitro studies to complete endpoint skin sensitization.
Qualifier:
according to
Guideline:
other: QSAR Prediction
Version / remarks:
In silico model DEREK NEXUS version 6.0.1.
Deviations:
no
Principles of method if other than guideline:
Thio Isocyanate Adduct consists of four constituents with the same functional groups and the functional groups are all substituted with CH2-carbon atoms, it is considered justified to extrapolate the DEREK prediction for constituent A to all four constituents.
GLP compliance:
not specified
Specific details on test material used for the study:
Test item identification: Thio Isocyanate Adduct.
Chemical name: Reaction product of 1-propanethiol, 3-(trimethoxysilyl)- and 1,6-diisocyanato-2,2,4(or 2,4,4)-trimethylhexane (1:1).
Molecular weight: 392.59.
Molecular formula: C16H32N2SSiO5
Key result
Parameter:
other: EC3 (%)
Remarks:
In silico prediction
Value:
>= 0.11
Remarks on result:
positive indication of skin sensitisation
Remarks:
Strong sensitizer
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Remarks:
Based on QSAR assessment.
Conclusions:
Thio Isocyanate Adduct is predicted to be sensitising to the skin with an EC3 of 0.11%.
Executive summary:

The objective of this assessment was to obtain a prediction on the potential for skin sensitisation of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 yielded an alert for constituent A from Thio Isocyanate Adduct for skin sensitisation based on the presence of an isocyanate (See attached report). Constituent A from Thio Isocyanate Adduct is predicted to be sensitising to the skin (plausible). DEREK NEXUS predicted an EC3 of 0.11% (strong sensitizer) for constituent A from Thio Isocyanate Adduct based on six closest structurally related substances. As the alert fired due to the isocyanate group and substituents on the carbons next to this group are not relevant, the result of constituent A can be extrapolated to Thio Isocyanate Adduct.

In conclusion, Thio Isocyanate Adduct is predicted to be sensitising to the skin with an EC3 of 0.11%.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 04 June 2018 and the experimental completion date was 08 June 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The assay results for SPCL were accepted although the standard deviation for the test item replicates for the Percent Lysine Depletion was 15.9% which is above the acceptance criterium of 11.6%. The variation in the depletion was most likely caused by bubbles in the sample which could not be removed. Since all replicates displayed significant depletion and the test item was classified in the “high reactivity class” even when only the lowest depletion results was used, this deviation did not affect the outcome of the study.
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Remarks:
The characterization of the test item was conducted under a sponsor quality system. Concentration, stability and homogeneity of the test item were not determined. The test item preparation was performed with approved procedures and documented in detail.
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Recommended test system in the international OECD guideline for DPRA studies.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BZA13264
- Expiration date of the lot/batch: 23 June 2018
- Manufacturing date: 23 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature under nitrogen
- Not stable at higher temperatures
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v) and dimethylsulfoxide (DMSO):ACN (1:9, v/v). At a concentration of 100 mM, Thio Isocyanate Adduct was not soluble in MQ and ACN:MQ (1:1, v/v), but was soluble in ACN, isopropanol, acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v). Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction for the purity/composition of the test item was performed. Based on the proportions of the main components of the test item together with their individual molecular weights, an apparent molecular weight of 589.02 g/mol was used for preparation of the test item stock solutions.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : For both the cysteine and lysine reactivity assay 91.89 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1560 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Details on study design:
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch: 110116HS_MHe_W0418.
- Positive control: Cinnamic aldehyde
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions. Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
- SPCC Calibration Curve. A SPCC calibration curve was prepared as described in Table 1.
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution. A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions. Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
- SPCL Calibration Curve. A SPCL peptide calibration curve was prepared as described in table 3.
- The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Tables 2 and 4.
- Sample incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The samples that showed a phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.
- HPLC-PDA Analysis. SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)
- Acceptability criteria. The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.
- Data Evaluation. The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= (1−(Peptide Peak Area in Replicate Injection (at 220 nm))/Mean Peptide Peak Area in Reference Controls (at 220 nm))x100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%- Data Interpretation. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table 5), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Positive control results:
Cysteine reactivity assay: The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.9% ± 1.4%.
Lysine reactivity assay: The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 48.5% ± 2.2%.
Key result
Parameter:
other: % SPCC Depletion
Run / experiment:
Cysteine Reactivity Assay
Value:
>= 96
Negative controls validity:
valid
Remarks:
The test item did not co-elute with SPCC
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: % SPCL Depletion
Run / experiment:
Lysine Reactivity Assay
Value:
>= 39.9
Negative controls validity:
valid
Remarks:
The test item did not co-elute with SPCL
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
- Cysteine reactivity: The mean peptide concentration of Reference Control A was 0.508 ± 0.009 mM while the mean peptide concentration of Reference Controls was 0.503 ± 0.001 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 18.67. The mean A220/A258 ratio ± 10% range was 16.80-20.54. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. Cysteine reactivity assay. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.9% ± 1.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
- Lysine reactivity: The mean peptide concentration of Reference Controls A was 0.525 ± 0.010 mM while the mean peptide concentration of Reference Controls C was 0.533 ± 0.016 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion. The CV of the peptide areas for the nine Reference Controls B and C was 3.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 14.43. The mean A220/A258 ratio ± 10% range was 12.98-15.87. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 48.5% ± 2.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Table 6. SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

SPCC Depletion (%)

PCcys-1

798268

0.134

71.5

PCcys-2

853969

0.45

69.5

PCcys-3

876813

0.149

68.7

-

-

Mean

69.9

-

-

SD

1.4

 

Table 7. SPCC Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test Item Samples

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

SPCC Depletion (%)

Peak area at 258 nm (µAU)

Area ratio (A220/A258)

209325/B-cys-1

100076

0.006

96.4

5000

20.02

209325/B-cys-2

99917

0.006

96.4

4701

21.25

209325/B-cys-3

136899

0.013

95.1

4256

32.16

-

-

Mean

96.0

N/A

24.48

-

-

SD

0.8

N/A

6.68

 

Table 8. SPCL Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

SPCC Depletion (%)

PClys-1

1099025

0.258

50.2

PClys-2

1123158

0.264

49.1

PClys-3

1191837

0.281

46.0

-

-

Mean

48.5

-

-

SD

2.2

 

Table 9. SPCL Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test Item Samples

Sample code

Peak area at 220 nm (µAU)

Concentration (mM)

SPCC Depletion (%)

Peak area at 258 nm (µAU)

Area ratio (A220/A258)

209325/B-lys-1

1023928

0.239

53.6

68145

15.03

209325/B-lys-2

1246430

0.294

43.6

83383

14.95

209325/B-lys-3

1711301

0.410

22.5

120883

14.16

-

-

Mean

39.9

N/A

14.71

-

-

SD

15.9

N/A

0.48

Interpretation of results:
study cannot be used for classification
Conclusions:
Thio Isocyanate Adduct was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of the study was to determine the reactivity of Thio Isocyanate Adduct towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures were based on OECD guideline 442C.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below:

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

> 0.99

0.994

> 0.99

0.997

Mean peptide concentration RC-A samples (mM)

0.50 +/- 0.05

0.508 +/- 0.009

0.50 +/- 0.05

0.525 +/- 0.010

Mean peptide concentration RC-C samples (mM)

0.50 +/- 0.05

0.503 +/- 0.001

0.50 +/- 0.05

0.533 +/- 0.016

CV(%) for RC samples B and C

< 15.0

1.1

< 15.0

3.2

Mean peptide depletion cinnamic aldehyde (%)

60.8 - 100

69.9

40.2 – 69.0

48.5

SD of peptide depletion cinnamic aldehyde (%)

< 14.9

1.4

< 11.6

2.2

SD of peptide depletion for the test item (%)

< 14.9

0.8

< 11.6

15.9

RC = Reference control; CV = Coefficient of Variation; SD = Standard Deviation

The following validation parameters were within the acceptability criteria for the DPRA: calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide SPCC depletion for the test item. The standard deviation value of the peptide SPCL depletion for the test item was outside the acceptability criteria for the DPRA. In the SPCL test item samples, a phase separation had occurred which most likely has affected the reproducibility of the sample injections. Since all other acceptability criteria were met, the DPRA results were accepted.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 96.0% SPCC depletion while in the lysine reactivity assay the test item showed 39.9% SPCL depletion. The mean of the SPCC and SPCL depletion was 67.9% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Test item

SPCC depletion (%)

SPCL depletion (%)

Mean of SPCC and SPCL depletion (%)

DPRA prediction and reactivity classification

Mean

SD

Mean

SD

Cysteine 1:10/Lysine 1:50 prediction model

Thio Isocyanate Adduct

96.0

0.8

39.9

15.9

67.9

Positive: High reactivity

In conclusion, this DPRA is considered to be valid. Thio Isocyanate Adduct was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 31 May 2018, and the experimental completion date was 22 June 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes
Remarks:
The characterization of the test item was conducted under a sponsor quality system. Concentration, stability and homogeneity of test item formulation were not determined. Test item preparation was performed with approved procedures and documented.
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
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).
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BZA13264
- Expiration date of the lot/batch: 23 June 2018
- Manufacturing date: 23 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature under nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated. A solubility test was performed. The test item was dissolved in DMSO to final concentrations of 40 mg/mL (clear colorless solutions). The 100-fold dilution of the 40, 20, 10 and 5 mg/mL DMSO stock in DMEM glutamax formed a homogeneous solution (slight precipitation). The 100-fold dilution of the 2.5 mg/mL DMSO stock formed a clear solution. The 400 μg/mL concentration (40 mg/mL stock) was selected as highest concentration for the main assay.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- No correction was made for the composition/purity of the test item.
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All
formulations formed a clear solution. Precipitation was observed at the start of the incubation period at dose levels of 200 μg/mL and upwards. At the end of the incubation period precipitation was observed at dose levels of 100 μg/mL and upwards and 200 μg/mL and upwards in the first and second experiment, respectively. Test item concentrations were used within 2.5 hours after preparation.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : diluted in DMSO.
Details on study design:
- Plating of Cells. For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+8 in experiment 1 and P+6 in experiment 2.
- Treatment of Cells. The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control 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±1.0ºC in the presence of 5% CO2. Initially, experiment 2 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.
- Luciferase Activity Measurement. The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (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 3 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 - 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
- Acceptability criteria. The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
- Data interpretation. A KeratinoSensTM 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 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 above 1.5 fold (i.e. at the EC1.5 determining concentration).
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW).
4. There is an apparent overall dose-response for luciferase induction.
Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.

Two independent experiments were performed. The cells were in these experiments incubated with Thio Isocyanate Adduct in a concentration range of 0.20 – 400 g/mL (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.
- Positive control: 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 4.5.1, 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.
- Vehicle control: The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
Positive control results:
In test 1, the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.75 and the EC1.5 59 μM.
In test 2, the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.18 and the EC1.5 18 μM.
Key result
Parameter:
other: Imax (µg/mL)
Run / experiment:
Experiment 1
Value:
>= 11.88
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax (µg/mL)
Run / experiment:
Experiment 2
Value:
>= 21.72
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 (µg/mL)
Run / experiment:
Experiment 1
Value:
>= 14
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 (µg/mL)
Run / experiment:
Experiment 2
Value:
>= 13
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (µg/mL)
Run / experiment:
Experiment 1
Value:
>= 75
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 (µg/mL)
Run / experiment:
Experiment 1
Value:
>= 87
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (µg/mL)
Run / experiment:
Experiment 2
Value:
>= 134
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 (µg/mL)
Run / experiment:
Experiment 2
Value:
>= 154
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Both 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 between 5 and 125 μM (59 μM and 18 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.75-fold and 4.18-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.5% and 7.0% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table 1. Overview luminescence induction and cell viability of test item

Conc. µg/mL

0.20

0.39

0.78

1.6

3.1

6.3

13

25

50

100

200

400

Exp. 1 Luminescence

1.18

1.21

1.27

1.46

1.36

1.41

1.39

2.13*

4.69*

11.88*

0.63

0.00

Exp. 1

Viability (%)

112.8

94.7

91.0

90.2

84.1

87.9

92.2

99.7

111.2

27.4

2.3

1.4

Exp. 2

Luminescence

1.16

1.04

1.16

1.13

1.06

1.18

1.49

2.16*

3.65*

21.72*

0.86

0.01

Exp. 2

Viability (%)

110.6

404.1

84.2

83.7

75.6

74.8

81.0

8.7

97.5

104.1

3.1

1.4

* P>0.001 Student’s test

Table 2. Overview luminescence induction and cell viability positive control EDMG

Conc. µg/mL

7.8

16

31

63

125

250

Exp. 1 Luminescence

1.11

1.19

1.23

1.54*

2.16*

3.75*

Exp. 1

Viability (%)

94.3

94.9

97.9

94.3

95.5

96.9

Exp. 2

Luminescence

1.36

1.46

1.8*

2.06*

2.95*

4.18*

Exp. 2

Viability (%)

100.3

104.8

101.1

109.8

120.8

106.4

* P>0.001 Student’s test

Table 3. Overview EC1.5, Imax, IC30 and IC50 values

 

EC1.5 (µg/mL)

Imax

IC30 (µM)

IC50 (µM)

Test item Exp. 1

14

11.88

75

87

Test item Exp. 2

13

21.72

134

154

Pos. control Exp. 1

59

3.75

N/A

N/A

Pos. control Exp. 2

18

4.18

N/A

N/A

N/A = Not applicable

Interpretation of results:
other: Thio Isocyanate Adduct is classified as positive.
Conclusions:
Thio Isocyanate Adduct is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions of this test.
Executive summary:

The study was carried out to evaluate the ability of Thio Isocyanate Adduct to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures were based on OECD guideline 442D and according to GLP. Thio Isocyanate Adduct was dissolved in dimethyl sulfoxide at 40 mg/mL. 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 0.20 - 400 μg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item precipitated at dose levels of 100 μg/mL and upwards and 200 μg/mL and upwards in the first and second experiment, respectively. Two independent experiments were performed. Both experiments passed the acceptance criteria. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Thio Isocyanate Adduct showed toxicity (IC30 values of 75 μg/mL and 134 μg/mL and IC50 values of 87 μg/mL and 154 μg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 14 μg/mL and 13 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 11.88-fold and 21.72-fold in experiment 1 and 2, respectively. Thio Isocyanate Adduct is classified as positive in the KeratinoSensTM assay 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 conclusion, Thio Isocyanate Adduct is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Endpoint:
skin sensitisation, other
Remarks:
Weight of Evidence
Type of information:
other: Weight of Evidence
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: ECHA Guidance on information requirements and chemical safety assessment
Version / remarks:
Chapter R.7a Endpoint specific guidance v.6.0 July 2017, paragraph 7.3.
Deviations:
no
Principles of method if other than guideline:
As the substance is a multiconstituent and not a UVCB the following strategy was followed:
STEP 0: The starting point for the weight of evidence is the assessment whether new studies are required. Available information on the test item was used to evaluate whether:
- The substance is not a strong acid (pH≤ 2.0) or base (pH ≥ 11.5), known to be not corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature.
- No adequate existing human data, which provide evidence that the substance is a skin sensitizer are available.
- No data from existing studies on skin sensitization in laboratory animals, which provide sound conclusive evidence that the substance is a sensitizer or non-sensitizer are available.
If no reliable data on solubility is available, a solubility test is performed to determine whether the substance dissolves sufficiently in a solvent which is appropriate for each test mentioned below. In case the solubility test demonstrates solubility of the test substance that meets the precondition limits for the in vitro tests, the following step-wise testing approach is followed:
STEP 1: DEREK assessment (overall skin sensitizing events), Direct Peptide Reactivity Assay (DPRA; molecular interaction with skin proteins) and KeratinoSensTM assay (inflammatory response in keratinocytes) are performed.
STEP 2: Depending on the outcome of the studies performed in STEP 1 and in absence of a definite conclusion on possible skin sensitization, the U-SENSTM assay is performed (key event: activation of dendritic cells).
STEP 3: Based on a weight of evidence of all available data on the test item related to skin sensitization, an argument is prepared to conclude on the classification for the substance or, if no conclusion can be drawn, to conclude on the performance of an in vivo skin sensitization study.
GLP compliance:
no
Type of study:
other: Weight of Evidence
Justification for non-LLNA method:
Sufficient information is available to meet the information requirements for skin sensitization of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification in accordance with Regulation (EC) No 1272/2008 (CLP) and related amendments. A weight of evidence approach according to Annex XI, sections 1.2-1.5, to the REACH Regulation is used.
Specific details on test material used for the study:
Not applicable
Positive control results:
Not applicable
Key result
Parameter:
other: EC3 (%)
Run / experiment:
DEREK Prediction
Value:
>= 0.11
Remarks on result:
positive indication of skin sensitisation
Remarks:
Based on the presence of an isocyanate
Key result
Parameter:
other: Peptide depletion (%)
Remarks:
Mean of SPCC and SPCL depletion
Run / experiment:
DPRA test
Value:
>= 67.9
Remarks on result:
positive indication of skin sensitisation
Remarks:
High reactivity class
Key result
Parameter:
other: Imax
Run / experiment:
KeratinoSens assay
Value:
>= 11.88 - <= 21.72
Remarks on result:
positive indication of skin sensitisation

DEREK NEXUS version 6.0.1 predicted Thio Isocyanate Adductto be sensitizing to the skin (plausible) based on the presence of an isocyanate. The EC3 was predicted to be 0.11%.

A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay Thio Isocyanate Adductwas dissolved in acetonitrile at 100 mM. No co-elution of the test item with SPCC or SPCL was observed.In the cysteine reactivity assay the test item showed 96.0% SPCC depletion while in the lysine reactivity assay the test item showed 39.9% SPCL depletion. The mean of the SPCC and SPCL depletion was 67.9% and as a result the test item was considered to be positive in the DPRA and classified in the “highreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

A valid KeratinoSens assay was performed according to OECD 442D and GLP. For the KeratinoSens assayThio Isocyanate Adductwas dissolved in DMSO to a final concentration of 40 mg/mL.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 0.20 - 400 µg/mL (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. The test item precipitated at dose levels of 100 µg/mL and above and 200 µg/mL and above in the first and second experiment, respectively. Two independent experiments were performed.

Thio Isocyanate Adduct showed toxicity (IC30 values of 75 µg/mLand 134 µg/mLand IC50 values of 87 µg/mL and 154 µg/mLin experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 14µg/mL and 13 µg/mLin experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 11.88-fold and 21.72-fold in experiment 1 and 2, respectively. Thio Isocyanate Adductis classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 200µg/mLwith a cell viability of >70% compared to the vehicle control.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on all available relevant information, including in silico/in chemico/in vitro data, Thio Isocyanate Adduct is classified as a skin sensitizer classified in category 1A.
Executive summary:

The objective of the study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including in silico/in chemico/in vitro data.

A DEREK assessment, DPRA assay and KeratinoSens assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

DEREK predicted Thio Isocyanate Adduct to be a skin sensitizer with an EC3 of 0.11%. The DPRA showed a mean of SPCC and SPCL depletion of 67.9% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Thio Isocyanate Adduct was classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mLwith a cell viability of >70% compared to the vehicle control.

Based on the above data and the knowledge that isocyanates are highly sensitizing, Thio Isocyanate Adduct is considered to be a skin sensitizer classified with category 1A. No further testing is required.

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

DEREK predicted the test substance to be a skin sensitizer with an EC3 of 0.11%. The DPRA showed a mean of SPCC and SPCL depletion of 67.9% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The test substance was classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 200µg/mLwith a cell viability of >70% compared to the vehicle control.

Based on the above data and the knowledge that isocyanates are highly sensitizing, the substance is considered to be a skin sensitizer classified with category 1A.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, The substance is classified a "Skin. Sens. 1A" with the hazard statement "H317: May cause an allergic skin reaction" according to Regulation (EC) No 1272/2008.

Based on cut-off values, registered substance is classified as "Resp. Sens. 1B" with the hazard statement "H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled" according to Regulation (EC) No 1272/2008.