Reaction product of trimethylhexamethylenediisocyanate and (3-mercaptopropyl)trimethoxysilane

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

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 reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guideline for DPRA studies.

Test material

Test material information

Constituent 1

Reference substance name:

2,4,4 TMDI function group 1 + mercaptan, mono-reacted

IUPAC Name:

2,4,4 TMDI function group 1 + mercaptan, mono-reacted

Constituent 2

Reference substance name:

2,4,4 TMDI function group 2 + mercaptan, mono-reacted

IUPAC Name:

2,4,4 TMDI function group 2 + mercaptan, mono-reacted

Constituent 3

Reference substance name:

2,2,4 TMDI function group 1 + mercaptan, mono-reacted

IUPAC Name:

2,2,4 TMDI function group 1 + mercaptan, mono-reacted

Constituent 4

Reference substance name:

2,2,4 TMDI function group 2 + mercaptan, mono-reacted

IUPAC Name:

2,2,4 TMDI function group 2 + mercaptan, mono-reacted

Constituent 5

Reference substance name:

2,2,4 bi-reacted compound

IUPAC Name:

2,2,4 bi-reacted compound

Constituent 6

Reference substance name:

2,4,4 bi-reacted compound

IUPAC Name:

2,4,4 bi-reacted compound

Constituent 7

Reference substance name:

2,4,4-trimethylhexa-1,6-diyl diisocyanate

EC Number:

239-714-4

EC Name:

2,4,4-trimethylhexa-1,6-diyl diisocyanate

Cas Number:

15646-96-5

Molecular formula:

C11H18N2O2

IUPAC Name:

1,6-diisocyanato-2,4,4-trimethylhexane

Constituent 8

Reference substance name:

2,2,4-trimethylhexa-1,6-diyl diisocyanate

EC Number:

241-001-8

EC Name:

2,2,4-trimethylhexa-1,6-diyl diisocyanate

Cas Number:

16938-22-0

Molecular formula:

C11H18N2O2

IUPAC Name:

1,6-diisocyanato-2,2,4-trimethylhexane

Test material form:

liquid

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.

In chemico test system

Details on the 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.

Results and discussion

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%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: % SPCC Depletion

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

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: % SPCL Depletion

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%).

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.