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

Endpoint summary

Description of key information

Testing for the skin sensitization endpoint was undertaken initially using in vitro / in chemico techniques.  The OECD 442C DPRA study was undertaken first, and indicated that the substance was a non-sensitiser.

The OECD 442D study was then undertaken, and again indicated that the substance was a non-sensitiser.  However, during this study, the results of the log Kow study became available, indicating that this value was > 6.5.  The OECD 442D study guidelines quotes as follows:

In general test substances with a LogP of up to 5 have been successfully tested whereas extremely hydrophobic substances with a LogP above 7 are outside the known applicability of the test method For test substances having a LogP falling between 5 and 7, only limited information is available.

On this basis, it was deemed that the results of the OECD 442D study were inconclusive.

Furthermore, consideration of the OECD 442E study states as follows:

Test chemicals with a Log Kow greater than 3.5 tend to produce false negative results. Therefore negative results with test chemicals with a Log Kow greater than 3.5 should not be considered.

On the basis that the data set for the substance is to be used for overseas notification, due consideration was therefore given to whether to conduct the OECD 442E study or to move to the OECD 429 LLNA study.  As the requirements for sensitization in REACH are the above 3 in Annex VII 8.3.1 are as follows:

— the available in vitro/in chemico test methods are not applicable for the substance or are not adequate for classification and risk assessment according to point 8.3.

An in vivo study shall be conducted only if in vitro/in chemico test methods described under point 8.3.1 are not applicable, or the results obtained from those studies are not adequate for classification and risk assessment according to point 8.3.

It was considered that the in vitro studies were not suitable for classification and risk assessment of this substance, and given that the LLNA study was required for other worldwide jurisdictions, the decision to proceed to this test was considered appropriate.

Skin Sensitisation - In Chemico

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.

Skin Sensitisation - In Vitro

Due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to an inconclusive outcome in the KeratinoSens assay.

Skin Sensitisation - In vivo (LLNA)

Non-sensitizer in female CBA/Ca mice.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

Specific details on test material used for the study:

No further details specified in the study report.

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.

Positive control results:

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

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.

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 May 2017 to 21 July 2017

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Under the experimental conditions of this study, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to a negative outcome in the KeratinoSens assay. However, it is worthy to note that the log P is > 6.5. According to the OECD Guideline 442D and in the context of an integrated approach to testing and
assessment, this information should be considered and the results of this test be taken with caution. Under the experimental conditions of this study, due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to an inconclusive outcome in the KeratinoSens assay.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

OECD Guideline 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, adopted on February 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Specific details on test material used for the study:

No further details specified in the study report.

Details on the study design:

PRINCIPLE OF THE ASSAY
This in vitro test uses Human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens is a stably transfected cell line with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.
Potential skin sensitizers are applied to the cells at 12 different concentrations for a period of 48 hours.
Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE-element of the human AKR1C2. The luciferase production is measured by flash luminescence.
In parallel, cytotoxicity is measured by a MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent runs.

Vehicle and negative control
Based on solubility results, the selected vehicle was DMSO.
This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium.
Since several test items were assayed concurrently, the results of the negative control were shared.

Positive control
Name: Cinnamic Aldehyde (CA)
Synonym: trans-Cinnamaldehyde
CAS number: 14371-10-9
Storage conditions: At +4 °C and under nitrogen gas
Since several test items were assayed concurrently, the results of the positive control were shared.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations.
Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM.
All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Test item formulations
On the basis of solubility results, the test item was dissolved in DMSO at 100 mM for the treatment of the first run. Since high cytotoxicity was observed in the first run, a pre-formulation at 100 mM was then diluted at 1 mM for the treatment of the second run.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of two for the first run or using a dilution factor of 1.41 for the second run (since high cytotoxicity was observed in the first run), to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provided by Givaudan.
Batch: C1.
Storage condition: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

STUDY DESIGN
The test item was tested in two independent runs using cells from a different passage number. The plates were processed as described in the Method.

Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).
Since the test item was found soluble in DMSO at 100 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 1000 μM. Then, a visual inspection of the sample was performed to evaluate the presence or absence of precipitate.

Method for a run of KeratinoSens assay
Cell seeding for testing
Cells were grown using general culture procedures up to 80-90% confluence,
the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
-after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Endpoint measurements
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates
After incubation, the supernatants from the white assay plates were discarded,
the cells were washed once with D-PBS,
a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
− 50 μL of the luciferase substrate was added to each well,
− 1 second after this addition, the luciferase signal was integrated for 2 seconds.

Absorbance signal to evaluate the cytotoxicity - transparent plate
For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
the plates were covered with a sealing membrane and returned at 37 °C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Run / experiment:

other: Mean of 2 runs

Parameter:

other: Viability & induction values

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'- thiodi-p-cresol, led to an inconclusive outcome

Other effects / acceptance of results:

SOLUBILITY TEST
In the solubility test, the test item was found not soluble in any recommended vehicles (i.e. DMSO, water for injections and culture medium) at 200 mM, even after 10 minutes of sonication and 10 minutes of heating at 60 °C.
However, the test item was found soluble at 100 mM in DMSO (without sonication or heating). Therefore, this vehicle (DMSO) was selected for the preparation of the test item stock formulations.
Precipitate was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 1000 μM.

KERATINOSENS RUN
At initiation of the present KeratinoSens study, the partition coefficient of the test item was unknown and was under determination at CiToxLAB Hungary. The Log P value was made available after the end of the experiment phase of the present study and the corresponding value was > 6.5. A test item with a partition coefficient greater than 5 cannot be concluded as negative with certainty in the KeratinoSens assay and results from both runs were analyzed accordingly.

First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.
This run was performed using the following concentrations 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO.
At these tested concentrations:
-a slight to strong precipitate was observed in test item-treated wells at concentrations ≥ 250 μM at the end of the 48-hour treatment period,
-a high decrease in cell viability (i.e. cell viability < 70%) was noted at dose levels ≥ 3.91 μM, except at 1000 μM where no cytotoxicity was noted. The slight to strong test item precipitation observed at dose levels ≥ 250 μM at the end of the 48-hour treatment period may have prevented the exposition of the cells to the test item. That may explain the absence of cytotoxicity at the highest dose level of 1000 μM. As a consequence, the viability values at dose levels ≥ 250 μM were not considered as biologically relevant and these dose levels were considered as highly cytotoxic. The corresponding IC30 and IC50 were thus not calculated but are considered to be between 1.95 and 3.91 μM,
-no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations showing a viability > 70%,
-the Imax was 1.67 but no EC1.5 was calculated in the absence of statistical significance,
The evaluation criteria for a negative response were met in this run. Since the log P is > 6.5, the conclusion on the lack of activity cannot be drawn with sufficient confidence (potential false negative results) and the results are considered inconclusive.

Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 14.66). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
This run was performed using the following concentrations 0.23, 0.32, 0.45, 0.64, 0.90, 1.27, 1.79, 2.53, 3.57, 5.03, 7.09, and 10 μM in culture medium containing 1% DMSO.
At these tested concentrations:
-no precipitate was observed in any test item-treated wells at the end of the 48-hour treatment period,
-a high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 5.03 μM,
-the corresponding IC30 and IC50 were calculated to be 4.53 and 5.14 μM, respectively,
-an apparent dose-response for luciferase induction was noted up to the cytotoxic dose levels. A statistically significant gene-fold induction above the threshold of 1.5 when compared to the negative control was only obtained for a highly cytotoxic concentration (i.e. 7.09 μM, with 5% viability), . the Imax was 2.71.
In this run, statistically non-significant inductions above 1.5-fold (i.e. inductions of 1.6 and 2.7 at concentrations of 3.57 and 5.03 μM, respectively) were followed by a higher concentration with a statistically significant induction (i.e. induction of 2.1 at 7.09 μM). Thus, the parameters used for the EC1.5 extrapolation were corrected manually using the formula described in the § Results analysis and was 3.22 μM.
Since the statistically significant induction above the threshold of 1.5 was obtained only for a cytotoxic concentration, this run is considered as negative. However, since the log P is > 6.5, the conclusion on the lack of activity cannot be drawn with sufficient confidence (potential false negative results) and the results are considered inconclusive.

Evaluation of the viability (%) of cultures treated with the test item for each run

	Concentrations (μM)
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	0.49	0.98	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000
Viability (%) in Run 1	119	174	177	8	0	0	0	1	2	9	28	99

 

	Concentrations (μM)
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	0.23	0.32	0.45	0.64	0.90	1.27	1.79	2.53	3.57	5.03	7.09	10
Viability (%) in Run 2	102	145	128	120	134	151	153	144	104	52	5	0

 

Gene induction values, I_max, IC₃₀, IC₅₀and EC_1.5values, mean and SD values obtained after treatment with the test item for each run

	Concentrations (μM)
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	0.49	0.98	1.95	3.91	7.81	15.6	31.3	63	125	250	500	1000
Induction values in Run 1	1.0	1.2	1.2	1.4	1.7	0.1	0.0	0.0	0.0	0.0	0.0	0.0

 

	Concentrations (μM)
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	0.23	0.32	0.45	0.64	0.90	1.27	1.79	2.53	3.57	5.03	7.09	10
Induction values in Run 2	1.0	0.7	0.8	1.0	1.3	1.3	1.3	1.3	1.6	2.7	2.1	1.4

 

I_maxand EC_1.5results

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	Imax	EC1.5 (μM)	IC50 (μM)	IC30 (μM)
Run 1	1.67	-	n.c.	n.c.
Run 2	2.71	3.22	5.14	4.53
Mean	2.19	n.r.	n.r.	n.r.
Geometric Mean	n.r.	n.c.	n.c.	n.c.
SD	0.74	n.c.	n.c.	n.c.

-: no data available

n.c.: not calculated

n.r.: not requested by the OECD Guideline

 

NOTE: the EC_1.5of the second run was manually calculated in agreement with the study plan.

 

Evaluation of the viability (%) of cultures treated with the positive control for each run

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64
Viability (%) in Run 1	108	115	101	138	109

 

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64
Viability (%) in Run 2	103	110	118	130	135

 

Gene induction values, I_max, IC₃₀, IC₅₀and EC_1.5values obtained with the positive control for each run

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64	Imax	EC1.5(μM)	IC50(μM)	IC30(μM)
Run 1	1.1	1.3	1.5	2.5	2.7	2.74	14.82	-	-

-: no data

 

	Concentrations (μM)
Cinnamic aldehyde	4	8	16	32	64	Imax	EC1.5(μM)	IC50(μM)	IC30(μM)
Run 2	1.4	1.6	2.0	3.5	14.7	14.66	6.09	-	-

-: no data

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of the study, due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to an inconclusive outcome in the KeratinoSens assay.

Executive summary:

The objective of the study was to evaluate the potential of the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

 

Results

At initiation of the present KeratinoSens study, the partition coefficient of the test item was unknown and was under determination at CiToxLAB Hungary. The Log P value was made available after the end of the experiment phase of the present study and the corresponding value was > 6.5. A test item with a partition coefficient greater than 5 cannot be concluded as negative with certainty in the KeratinoSens assay and results from both runs were analyzed accordingly.

 

First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

 

This run was performed using the following concentrations 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO.

 

At these tested concentrations:

-a slight to strong precipitate was observed in test item-treated wells at concentrations ≥ 250 μM at the end of the 48-hour treatment period,

-a high decrease in cell viability (i.e. cell viability < 70%) was noted at dose levels ≥ 3.91 μM, except at 1000 μM where no cytotoxicity was noted. The slight to strong test item precipitation observed at dose levels ≥ 250 μM at the end of the 48-hour treatment period may have prevented the exposition of the cells to the test item. That may explain the absence of cytotoxicity at the highest dose level of 1000 μM. As a consequence, the viability values at dose levels ≥ 250 μM were not considered as biologically relevant and these dose levels were considered as highly cytotoxic. The corresponding IC30 and IC50 were thus not calculated but are considered to be between 1.95 and 3.91 μM,

-no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations showing a viability > 70%,

-the Imax was 1.67 but no EC1.5 was calculated in the absence of statistical significance.

 

The evaluation criteria for a negative response were met in this run. However, since the log P is > 6.5, the conclusion on the lack of activity cannot be drawn with sufficient confidence (potential false negative results) and the results are considered inconclusive.

 

Second run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

 

This run was performed using the following concentrations 0.23, 0.32, 0.45, 0.64, 0.90, 1.27, 1.79, 2.53, 3.57, 5.03, 7.09, and 10 μM in culture medium containing 1% DMSO.

 

At these tested concentrations:

-no precipitate was observed in any test item treated wells at the end of the 48-hour treatment period,

-a high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 5.03 μM,

-the corresponding IC30 and IC50 were calculated to be 4.53 and 5.14 μM, respectively,

-an apparent dose-response for luciferase induction was noted up to the cytotoxic dose levels. A statistically significant gene-fold induction above the threshold of 1.5 when compared to the negative control was only obtained for a highly cytotoxic concentration (i.e. 7.09 μM, with 5% viability),

-the Imax was 2.71.

 

In this run, statistically non-significant inductions above 1.5-fold (i.e. inductions of 1.6 and 2.7 at concentrations of 3.57 and 5.03 μM, respectively) were followed by a higher concentration with a statistically significant induction (i.e. induction of 2.1 at 7.09 μM). Thus, the parameters used for the EC1.5 extrapolation were corrected manually using the formula described in the § Results analysis and was 3.22 μM.

 

Since the statistically significant induction above the threshold of 1.5 was obtained only for a cytotoxic concentration, this run is considered as negative. However, since the log P is > 6.5, the conclusion on the lack of activity cannot be drawn with sufficient confidence (potential false negative results) and the results are considered inconclusive.

 

Discussion

Under the experimental conditions of this study, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to a negative outcome in the KeratinoSens assay. However, it is worthy to note that the log P is > 6.5.

According to the OECD Guideline 442D and in the context of an integrated approach to testing and assessment, this information should be considered and the results of this test be taken with caution.

 

Conclusion

Under the experimental conditions of this study, due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to an inconclusive outcome in the KeratinoSens assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2017 to 21 November2017

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:

OECD Guidelines for Testing of Chemicals No. 429 Skin Sensitisation: Local Lymph Node Assay (22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. Skin Sensitisation: Local Lymph Node Assay (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further information specified in the study report.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 10 weeks old (age-matched, within one week)
Body weight range at starting: 21.6 – 23.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatisation time: 21 days
Note: In the Preliminary Experiment, mice of 10 weeks of age (18.1-19.7 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.0 – 24.3 °C
Relative humidity: 23 - 79%
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 262 21592, Expiry date: 31 January 2018) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60163330050101, Expiry date: 28 November 2017 and Batch Number: 60172230020101, Expiry date: 11 August 2018, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary).

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50, 25 and 10 % (w/v).

No. of animals per dose:

4 animals per dose

Details on study design:

Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50 and 25 % (w/v). The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).
In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored using Table 2 [4]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. Test item precipitation or minimal amount of test item precipitation was observed from Day 1 to Day 3 for both of the animals in the 50 % (w/v) group. There were no indications of any irritancy at the site of application.
No mean marked body weight loss (>5% reduction of body weight) was observed in any of the groups.
Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6.
The ear thickness values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).

Based on these observations, 50 % (w/v) dose was selected as top dose for the main test.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath.
Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
At the radio-label counting stage, there was a higher degree of fluctuations than usual in the signals of some samples, at the time of the measurement indicated in the Study Plan. In order to be sure of adequate data, the whole set of samples (including the background vials and the samples of the negative and positives control groups) were re-measured 4 times (within 2 days after the preparation) to produce proper data. Based on the half-life of the 3H isotope (over 12 years), this difference would not influence the activity of the samples in a measurable manner. In the report, the DPM values from the 4th run are shown, but all the data of the individual measurements are kept and archived in the raw data binder. This fact was considered not to adversely affect the results or integrity of the study.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not specified.

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 3.3) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

50% (w/v)

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25% (w/w)

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10% (w/w)

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. No test item precipitation was observed in the study. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
No marked body weight losses (≥5%) was observed on the mean body weight changes in any groups. However, marked body weight loss (>5%) was detected for one animals in the 50 % (w/v) dose group. This difference is considered to be individual variability, and not related to the test item.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in the 50, 25 and 10 % (w/v) test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.
The stimulation index values were 1.2, 1.1 and 1.1 at concentrations of 50, 25 and 10 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was a white powder, which was formulated in AOO. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change^#(%)
5059 5065 5056 5068	1 2 3 4	Negative (vehicle) control in AOO     Mean	23.9 22.2 22.3 21.6 22.5	23.2 21.6 23.6 20.8 22.3	-2.9 -2.7 5.8 -3.7 -0.9
5060 5069 5071 5064	5 6 7 8	6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 50% (w/v) in AOO     Mean	21.9 23.7 22.9 22.5 22.8	22.0 22.5 22.1 22.6 22.3	0.5 -5.1 -3.5 0.4 -1.9
5074 5057 5072 5066	9 10 11 12	6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 25% (w/v) in AOO     Mean	22.3 22.5 22.7 21.7 22.3	23.7 23.6 22.5 21.5 22.8	6.3 4.9 -0.9 -0.9 2.3
5062 5075 5063 5073	13 14 15 16	6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 10% (w/v) in AOO     Mean	23.0 22.0 22.4 21.7 22.3	25.4 23.2 22.1 21.9 23.2	10.4 5.5 -1.3 0.9 3.9
5058 5061 5067 5070	17 18 19 20	Positive control 25% (w/v) HCA in AOO     Mean	22.7 22.8 22.8 21.6 22.5	22.0 21.9 22.3 23.7 22.5	-3.1 -3.9 -2.2 9.7 0.1

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group †	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	32	-	-	-	-
Negative control (AOO)	7595	7563.0	8	945.4	1.0
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 50% (w/v) in AOO	8804	8772.0	8	1096.5	1.2
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 25% (w/v) in AOO	8690	8658.0	8	1082.3	1.1
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 10% (w/v) in AOO	8524	8492.0	8	1061.5	1.1
Positive control (25% (w/v) HCA in AOO)	25331	25299.0	8	3162.4	303

Note:

1. † = For three samples, the radioactivity counter could not resolved the count in one measurement run (probably due to quenching). As per standard practice, consequent runs were made, and the result if the first complete run (the fourth) are presented above.

 

Summarized Clinical Observations

Group	Identity No.	CLINICAL OBSERVATIONS
		DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (AOO)	1   2   3   4	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 50% (w/v) in AOO	5   6   7   8	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 25% (w/v) in AOO	9   10   11   12	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 10% (w/v) in AOO	13   14   15   16	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
Positive control (25% (w/v) HCA in AOO)	17   18   19   20	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free

Note:

1.BT: before treatment, AT: after treatment

 

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2016)

CBA/CaOlsHsd mice
	Vehicles
	Acetone : Olive oil 4:1 (AOO)			1% Pluronic PE9200 in water (1% Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	549.4	3609.5	7.9	214.5	1890.2	9.9
Range:min            max	111.3 1990.1	890.3 7674.5	3.3 18.4	23.0 680.8	154.0 6755.8	3.0 33.1
Number of cases	49	53	51	153	145	137

 

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	253.5	2562.4	10.5	474.9	3053.9	7.5
Range:min            max	94.8 649.6	1201.3 4804.6	4.9 21.3	224.36 934.6	1083.8 4877.5	3.1 14.5
Number of cases	34	30	30	19	19	17

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	235.4	2371.8	10.0	303.7	4922.3	17.7
Range:min            max	63.3 506.0	817.3 4978.0	6.5 14.4	183.5 489.6	2456.3 8682.5	8.9 36.3
Number of cases	14	14	14	12	13	13

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the assay, 6,6’-di-tert-butyl-2,2’-thiodip-cresol, tested in a suitable vehicle, was shown to have no skin sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
The following classification/labelling is triggered: none.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol following dermal exposure in mice. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the best vehicle for the test item was acetone:olive oil 4:1 (v:v) mixture (AOO). The 50 % (w/v) formulation was the highest concentration which was suitable for the test. The formulations appeared to be solutions by visual examination.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 % (w/v) and 25 % (w/v) in AOO. Based on the observations recorded in the preliminary test, 50 % (w/v) was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (formulated in AOO) at 50, 25 and 10 % (w/v) concentrations respectively,

- the negative control group received the vehicle (AOO) only,

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or signs of systemic toxicity were observed during the study. No test item precipitation was observed in the study. There were no indications of any irritancy at the site of application.

 

No marked body weight losses (≥5%) was observed on the mean body weight changes in any groups. However, marked body weight loss (>5%) was detected for one animals in the 50 % (w/v) dose group. This difference is considered to be individual variability, and not related to the test item.

 

The stimulation index values were 1.2, 1.1 and 1.1 at concentrations of 50, 25, and 10 % (w/v), respectively.

 

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, 6,6’-di-tert-butyl-2,2’-thiodip-cresol, tested in a suitable vehicle, was shown not to have a skin sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

The following classification/labelling is triggered: none.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensistation - In Chemico

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides.  

Results

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.

Skin Sensitisation - In Vitro

The objective of the study was to evaluate the potential of the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, to activate the Nrf2 transcription factor.

Results

Under the experimental conditions of the study, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to a negative outcome in the KeratinoSens assay. However, it is worthy to note that the log P is > 6.5.

According to the OECD Guideline 442D and in the context of an integrated approach to testing and assessment, this information should be considered and the results of this test be taken with caution.

Conclusion

Under the experimental conditions of this study, due to its log P > 6.5, the test item, 6,6'-di-tert-butyl-2,2'-thiodi-p-cresol, led to an inconclusive outcome in the KeratinoSens assay.

Skin Sensitisation - In Vivo (LLNA)

The aim of this study was to determine the skin sensitisation potential of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol following dermal exposure in mice. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

Results

No mortality or signs of systemic toxicity were observed during the study. No test item precipitation was observed in the study. There were no indications of any irritancy at the site of application. 

No marked body weight losses (≥5%) was observed on the mean body weight changes in any groups. However, marked body weight loss (>5%) was detected for one animals in the 50 % (w/v) dose group. This difference is considered to be individual variability, and not related to the test item.

The stimulation index values were 1.2, 1.1 and 1.1 at concentrations of 50, 25, and 10 % (w/v), respectively.

Conclusion

Under the conditions of the present assay, 6,6’-di-tert-butyl-2,2’-thiodip-cresol, tested in a suitable vehicle, was shown not to have a skin sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was negative in both a OECD 442C and LLNA assay. No classification is therefore applicable

in accordance with Regulation (EC) 1272/2008.