6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2017 to 24 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

OECD guideline No. 442C: in chemico skin sentitization: Direct Peptide Reactivity Assay (DPRA), adopted on 04 February 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

EC Number:

202-009-7

EC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Cas Number:

90-66-4

Molecular formula:

C22H30O2S

IUPAC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Test material form:

solid: particulate/powder

Details on test material:

Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Other name: LOWINOX® TBP-6
CAS number: 90-66-4
Batch/Lot Number: C034J0059 / C036K0111
Description: White powder
Purity*: 99.8%
Expiry date: 12 May 2017
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
* No correction for purity of the test item was applied.

Specific details on test material used for the study:

No further details specified in the study report.

In chemico test system

Details on the study design:

PRINCIPLE OF THE ASSAY
The assay determines the chemical reactivity of the test item to cysteine and lysine peptides, which is a unifying characteristic of most skin sensitizing chemicals (Gerberick et al. 2004, Gerberick et al. 2007). Reactivity (% depletion) is determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with Ultra-Violet detection. Complete details of the analysis process are described in CiToxLAB France analytical method procedure "A LC/UV analytical method for the determination of Direct Peptide Reactivity".
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:

%depletion = [1- (Peptide Peak Area in Replicate Injection/Mean Peptide Area in relevant Reference Control C samples)] x 100

Vehicle
Based on solubility results, the retained vehicle was acetonitrile.
As several test items were assayed concurrently with this vehicle, its results were shared.
Positive control
The positive control was cinnamaldehyde (CAS No. 104-55-2), batch No. MKBV4784V, supplied by Sigma-Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%.
As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was dissolved in acetonitrile at 100 mM. The physical aspect of the formulation was a colorless liquid. The formulation was used just after its preparation.

Co-elution control samples
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent specified in § Reference control samples preparation. These samples were used to:
-reference control A: check the accuracy of the calibration curve for peptide quantification,
-reference control B: check the stability of the peptide during analysis,
-reference control C: check that the solvent did not impact the percentage of peptide depletion.

Test item formulation preparation
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation had the aspect of a colorless liquid. The formulation was used just after its preparation.

TEST SYSTEMS
Cysteine peptide
-Peptide sequence: Ac-RFAACAA-COOH
-Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
-Molecular weight: 750.88 g/mol
-Supplier: JPT Peptide Technologies GmbH
-Batch No.: 111016HS_MHeW0117
-Storage condition: At -20°C
-Description: White powder

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in CiToxLAB France files.
The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution. The detailed preparation method is described in a CiToxLAB France analytical method, specific to the DPRA test.

Lysine peptide
-Peptide sequence: Ac-RFAAKAA-COOH
-Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
-Molecular weight: 775.91 g/mol
-Supplier: JPT Peptide Technologies GmbH
-Batch No.: 220114HSDWW0117
-Storage condition: At -20°C
-Description: White powder

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in CiToxLAB France files.
The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution. The preparation method is described in a CiToxLAB France analytical method, specific to the DPRA test.

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.

Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

Co-elution control samples preparation
For the co-elution control with cysteine peptide:
50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide:
In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation
Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide:
50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide:
In parallel, 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Test item samples preparation
For the reactivity of test item with cysteine peptide:
50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide:
In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation (see § Results).
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
-one blank sample (peptide dilution buffer),
-one calibration curve injected at the beginning of the analytical batch,
-three reference control A samples,
-the co-elution control sample,
-three reference control B samples,
-reference control C sample (replicate 1),
-positive control sample (replicate 1),
-test item study sample (replicate 1).

The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
-three reference control B samples.

DATA ANALYSIS AND CALCULATION
Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test item samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test item replicate, the percent depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:

%depletion = [1-(Peptide Peak Area in Replicate Injection / Mean Peptide Peak Area in relevant Reference Control C sample)] x 100

Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

Evaluation of the possible co-elution of the test item with the lysine or cysteine peptides
In order to detect possible co-elution of the test items with a peptide, chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

Results and discussion

Positive control results:

Reported in table form - See "Any other information" for details.

In vitro / in chemico

Results

Key result

Parameter:

other: mean of the percent cysteine and percent lysine depletions

Value:

6.38

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples (Figures 1 and 4) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:
-for the cysteine peptide, the mean depletion value was 0.04%,
-for the lysine peptide, the mean depletion value was 0.00%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.02%. However, since precipitate was observed at the end of the incubation with the peptides, the peptides depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no or minimal reactivity. Therefore, the DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Any other information on results incl. tables

Percent peptide depletion for the test item samples

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE DEPLETION IN SAMPLES SPIKED WITH A

SOLUTION AT 100 mM OF LOWINOX TBP-6

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Lowinox TBP-6	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1 2 3	2530882 2504441 2498480	0.00^* 0.00^* 0.12	2362638 2386647 2345628	0.00^* 0.00^* 0.00^*
Mean SD % CV	- - -	0.04 0.07 173.2	- - -	0.00^* nc nc	0.02	No reactivity/ minimal reactivity
Precipitate:	Yes		Yes
Micelle	No		No

^*: Value set to 0 due to negative depletion

-: not applicable

nc: not calculated

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE CONCENTRATION IN REFERENCE

CONTROL C SAMPLES PREPARED IN ACETONITRILE DEGAZEE

Sample number	Cysteine peptide			Lysine peptide
	Peak area (μV/sec)	Concentration (mM)	%Dev	Peak Area (μV/sec)	Concentration (mM)	%Dev
1 2 3	2532711 2470468 2501550	0.474 0.462 0.468	(-5.2) (-7.5) (-6.4)	2348303 2313793 2357013	0.497 0.490 0.499	(-0.5) (-2.0) (-0.2)
Mean SD % CV	2501576 - -	0.468 0.06 1.2	(-6.4) - -	2339703 - -	0.495 0.005 1.0	(-0.9) - -

 

DETERMINATION OF % INTERFERENCE DUE TO CO-ELUTION OF LOWINOX TBP-6 WITH

CYSTEINE OR LYSINE PEPTIDES

	Peak detected at the cysteine retention time		Peak detection at the lysine retention time
Sample number	Peak Area (μV/sec)	% Interference	Peak Area (μV/sec)	% Interference
1	0	(0.0)	0	(0.0)
Precipitate:	Yes		Yes
Micelle:	No		No

 

 

Percent peptide depletion for the positive control samples

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE DEPLETION IN SAMPLES SPIKES WITH A

SOLUTION AT 100 mM OF CINNAMALDEHYDE

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Cinnamaldehyde	Depletion classification
	Peak Area (μV/sec)	% depletion	Peak Area (μV/sec)	% depletion
1 2 3	85404 102814 93973	96.59 95.89 96.24	822945 869737 884959	64.83 62.83 62.18
Mean SD % CV	- - -	95.24 0.35 0.4	- - -	63.28 1.38 2.2	79.76	High reactivity

 

DETERMINATION OF CYSTEINE PEPTIDE AND LYSINE PEPTIDE CONCENTRATION IN REFERENCE

CONTROL C SAMPLES PREPARED IN ACETONITRILE

	Cysteine peptide			Lysine peptide
Sample number	Peak Area (μV/sec)	Concentration (mM)	%Dev	Peak Area (μV/sec)	Concentration (mM)	%Dev
1 2 3	2532711 2470468 2501550	0.474 0.462 0.468	(-5.2) (-7.5) (-6.4)	2348303 2313793 2357013	0.497 0.490 0.499	(-0.5) (-2.0) (-0.2)
Mean SD % CV	2501576 - -	0.468 0.006 1.2	(-6.4) - -	2339703 - -	0.495 0.005 1.0	(-0.9) - -

-: not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the peptides.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

Methods 

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was dissolved at 100 mM in acetonitrile.

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

-for the cysteine peptide, the mean depletion value was 0.04%,

-for the lysine peptide, the mean depletion value was 0.00%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 0.02%. However, since precipitate was observed at the end of the incubation with the peptides, the peptides depletion may be underestimated.

 

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item was considered to have no or minimal reactivity. Therefore, the DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

 

Conclusion

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the peptides.