(R*,R*)-1,4-dimercaptobutane-2,3-diol

Endpoint summary

Description of key information

• QSAR DEREK (in chemico): Skin sensitation is plausible for alert 432 (thiol or thiol exchange agent). Substance is predicted to be a skin sensitizer
• DPRA (OECD442C, in vitro): Inconclusive.
• LLNA (OECD 429, in vivo): Negative, non-sensitizer. Observed SI-values were 1.2, 0.8, 0.9 and 1.1 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April - 6 May 2020

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:

Adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Official Journal of the European Union L 142 of 31 May 2008 amendment L193/3 20 July 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90
- Sex: Females nulliparous and non-pregnant
- Age at study initiation: Young adult mice;12 weeks old
- Weight at study initiation: 20.0 - 22.9 g
- Housing:
During acclimatization period: Grouped caging in small groups
During the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
- Diet (e.g. ad libitum): ssniff® Rat/Souris-Elevage E complete diet
- Water (e.g. ad libitum): tap water
- Acclimation period: 14 days
- Indication of any skin lesions: No.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

1 %, 0.5 %, 0.25 % and 0.1 % (w/v)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
VEHICLE SELECTION
Preliminary test item formulation evaluation was performed following the recommendation of the relevant guidelines. Solubility of the test item was evaluated in the following vehicles (in order of preference). Concentration series of 100 %, 50 % etc. was used according to the guidelines.
1. Acetone: Olive oil 4:1 (v/v) mixture (AOO)
2. N,N-Dimethylformamide (DMF)
3. Dimethyl sulfoxide (DMSO)
4. Methyl ethyl ketone (MEK)
5. Propylene glycol (PG)
6. Purified water
The test item was soluble in all evaluated vehicle. The maximum soluble concentration was 100 % (w/v; i.e. 1 g/mL) in DMF using mechanical agitation and ultrasonic dispersion. In the other vehicles the maximum of 50 % (w/v) was achieved. Based on this and in accordance with the guideline DMF was selected as vehicle of the test item. The following vehicle was used in the test: N,N-Dimethylformamide (DMF)

NEGATIVE CONTROLS
- Vehicle control for the test item: The vehicle control group animals (used as negative control for the groups treated with the test item formulations) were treated with DMF concurrent to other treatment groups.
- Vehicle control for the positive control: The vehicle control group animals (used as negative control for the group treated with the positive control) were treated with AOO concurrent to other treatment groups.

POSITIVE CONTROL
Positive control group animals were treated with a-Hexylcinnamaldehyde (HCA) concurrent to test item treatment groups.
Name: a-Hexylcinnamaldehyde-technical grade, 85 %
Batch No.: MKCF6971
CAS Number: 101-86-0
Description: Light yellow liquid
Purity: 97.2 % (GC)
Expiry date: September 02, 2020
Supplier: Sigma-Aldrich
Storage condition: room temperature

DOSE RANGE FINDING TESTS (DFR):
The pre-experiments on formulation evaluation and the dose range finding tests (DRFs) were not performed in compliance with the GLP-Regulations and are excluded from the Statement of the Study Director, but the raw data of these tests are archived under the study code of the present study.
Two consecutive DRFs were performed. The DRFs were conducted in a similar experimental manner to the exposure phase of the main test except that there was no assessment of lymph node proliferation and fewer animals were used. The test item was formulated in DMF and evaluated at concentrations of 100 %, 50 %, 25 % and 10 % (w/v) in the first DRF, while 5 %, 2.5 % and 1 % (w/v) formulations were tested in the second DRF. Groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6, where applicable).
According to this 1 % (w/v) was used as the maximum concentration in the main test with the aim of testing the highest possible non-toxic, non-irritant concentration.
- Ear thickness measurements: Measurement of ear thickness was taken (where applicable) using digital micrometer on Day 1 (pre-dose), Day 3 (prior to the treatment) and Day 6.
- Systemic toxicity and Erythema scores: Both ears of each mouse were observed for erythema and scored according to criteria. In the first DRF mortality was observed in the 100 % (w/v) dose group (1 of the 2 animals) on Day 3. Significant symptoms of systemic toxicity and irritation were observed for the other animal in this dose group hence this animal was humanely killed on Day 4. Symptoms of a systemic effect and/or signs of a significant irritation (indicated by an erythema score = 3 and/or significantly increased ear thickness values*) were observed also in the other test groups (50 %, 25 % and 10 %; w/v).
In the second DRF no mortality or systemic toxicity (indicated by body weight loss > 5 % or other symptoms) were observed. Signs of irritation (loss of hair and/or scar) was observed in the 5 % and 2.5 % (w/v) dose groups (2 of the 2 animals in both groups; first observation was on Day 4) however no erythema sored as = 3 or significantly increased ear thickness values* were observed. Both animals were symptom-free during the whole test in the 1 % (w/v) dose group.
(* i.e. > 25 % increase compared to the initial value)


MAIN STUDY

MAIN TEST DESIGN
- Concentrations: The test item was administered at four different concentrations: 1, 0.5, 0.25 and 0.1 (w/w) . For test groups in the main test see below.
- Randomization; The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.

TREATMENT PREPARATION AND ADMINISTRATION:
- in vivo treatment: Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. 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: No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.

- Injection of 3HTdR: On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing 25 µCi# of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
# Remark: Breaking down of [methyl-3H]-Thymidine in aqueous solution (about 5 % per month) was taken into account as necessary when 3HTdR solution was prepared.

- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish 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 lymph node cells (LNCs), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

- Determination of Incirporated 3HTdR: After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants, then the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the ß-scintillation counter.
3HTdR incorporation was measured for up to 10 minutes per sample.# The ß-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA. Instrument used for the measurement:
Name: Tri-Carb 2900TR
Liquid Scintillation Analyzer
Serial Number: 427878
# Remark: The samples were not specifically stored. On the other hand, the samples were loaded into the LSC on May 05, 2020 and measured on May 06, 2020 only due to technical reason.

CLINICAL OBSERVATIONS
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test (according to criteria depicted in Table 2) in all test groups. Ear thickness was also measured in the following test groups: vehicle control for the test item (DMF, all test item treated groups (1 %, 0.5 %, 0.25 % and 0.1 %; w/v). Individual records were maintained for all observations.

INTERPRETATION OF THE RESULTS
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI = 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Positive control substance(s):

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

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
A significant lymphoproliferative response (SI = 3) was noted for HCA (SI = 9.4). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

Parameter:

SI

Value:

1.2

Test group / Remarks:

test item concentration of 1 % (w/v)

Parameter:

SI

Value:

0.8

Test group / Remarks:

test item concentration of 0.5 % (w/v)

Parameter:

SI

Value:

0.9

Test group / Remarks:

test item concentration of 0.25 % (w/v)

Parameter:

SI

Value:

1.1

Test group / Remarks:

test item concentration of 0.1 % (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No significant lymphoproliferative response (SI = 3) compared to the relevant vehicle control (DMF) was noted for 1,4-Dithiothreitol at the tested concentrations. No dose-related response was observed.

CLINICAL OBSERVATIONS:
Normal for all groups

BODY WEIGHTS
Normal for all groups

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay, 1,4-Dithiothreitol (CAS 3483-12-3) tested at the maximum feasible non-toxic, non-irritant concentration of 1 % (w/v) and also at concentrations of 0.5 %, 0.25 % or 0.1 % (w/v) as adequate homogeneous formulations (solutions, prepared with N,N-Dimethylformamide as vehicle) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of 1,4-Dithiothreitol (CAS 3483-12-3) following dermal exposure in the Local Lymph Node Assay.

Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Based on the solubility the maximum applicable concentration was 100 % (i.e. 1 g/mL) in N,N-Dimethylformamide (DMF). Significant adverse effects (systemic toxicity and/or irritation) were observed in the dose range finding tests (DRFs) at 2.5 % (w/v) and above. According to this the test item was examined in the main test as 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) formulations (apparently solutions) in DMF.

An appropriate positive control (a-Hexylcinnamaldehyde, HCA), and further two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 9.4) thus confirming the validity of the assay.

No mortality or signs of systemic toxicity were observed during the test. No significant, treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.

No significantly increased lymphoproliferation (indicated by an SI = 3) compared to the relevant control (DMF) was noted for 1,4-Dithiothreitol at the applied test concentrations. The observed stimulation index values were 1.2, 0.8, 0.9 and 1.1 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.61, r2 = 0.15; evaluated by linear regression using SI values).

According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI = 3) up to the maximum attainable non-toxic, non-irritant concentration of 1 % (w/v) as well as the lack of a significant dose-related response are considered as evidence that 1,4-Dithiothreitol is not a skin sensitizer.

In conclusion, under the conditions of the present assay, 1,4-Dithiothreitol (CAS 3483-12-3) tested at the maximum feasible non-toxic, non-irritant concentration of 1 % (w/v) and also at concentrations of 0.5 %, 0.25 % or 0.1 % (w/v) as adequate homogeneous formulations (solutions, prepared with N,N-Dimethylformamide as vehicle) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

other information

Study period:

11 February - 25 March 2020

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:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO Guideline 3030/99

Version / remarks:

SANCO Guideline 3030/99. Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414
rev.4, July 11, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol 154 Direct Peptide Reactivity assay (DPRA) for skin sensitisation testing

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch-No.: 44606300 / 50774
Storage: Refrigerator, 2-8°C, under Nitrogen

Details on the study design:

Principle of the DPRA Method
The reactivity of a test chemical and synthetic Cysteine or Lysine containing peptides is evaluated by combining the test chemical with a solution of the peptide and monitoring the remaining concentration of the peptide following 24 hours of interaction time at room temperature. The peptide is a custom m
aterial containing phenylalanine to aid the detection and either Cysteine (“C”) or Lysine (“K”) as the reactive center.
Relative concentrations of the peptides following the 24 hour incubation are determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analysed in triplicates in batches of up to 26 chemicals (including controls).

Steps of the DPRA Method done in chronological order
- Solubility assessment of the test chemical – ultrapure water was used as a solvent
- Preparation of buffer solutions
- Pre-weighting of test chemicals and positive control
- Pre-weighting of cysteine or lysine peptide for the stock solution
- Test chemical and positive control solution preparation
- Peptide stock solution preparation
- Serial dilution of standards
- Assembling of standards, reaction samples, positive controls, reference controls (A, B and C) and co-elution controls. For each set of control/sample replicates, the triplicate vials are prepared individually but from the same solutions.
- Preparation of HPLC system (column equilibration)
- HPLC analysis
- Data evaluation

Assembly of reaction samples and controls
1:10 ratio cysteine peptide
0.5 mM peptide, 5 mM test item
750 µL cysteine peptide stock solution (or phosphate buffer for the co-elution control)
200 µL acetonitrile50 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)

1:50 ratio lysine peptide
0.5 mM peptide, 25 mM test item
750 µL lysine peptide stock solution (or ammonium acetate buffer for the co-elution control)
250 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)

The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5°C - 30°C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches were consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended. The total HPLC analysis time should be less than 21 hours.

Formulation of the Test Item
The solubility of the test item was tested in a non-GLP preliminary solubility test as follows: the solubility of the test item was first evaluated at the concentration of 100 mM in acetonitrile. Acetonitrile dissolved the test item completely, a homogenous and clear solution was formed.
Next, the behavior of the formulation of the test item with the acetonitrile was determined in the buffers of the test system (phosphate and acetate buffer – 5.5) in a ratio corresponding to the reaction sample assembly (6.2). The compatibility of the formulation with the buffers of the test system was proven, no precipitate was observed in any cases and homogenous, clear solutions were obtained. Since this solvent is the preferred vehicle in the order of solvents listed in the guideline [3] and the formulation complied with all obligations of the test guideline, no more formulations were checked and acetonitrile was chosen as solvent.

Synthetic Peptides
Cysteine peptides
Name: Cysteine peptide
Batch no: 111016HSMHe-W0220-02
Storage: at -20°C or below
Purity: 90.64 %
Molecular weight: 750.88 g/mol
Sequence: Ac-RFAACAA-OH
Expiry date: August 25, 2020
Supplier: JPT Peptide Technologies GmbH

Name: Cysteine peptide
Batch no: 211019HS-MHe-2
Storage: at -20°C or below
Purity: 95.6 %
Molecular weight: 750.88 g/mol
Sequence: Ac-RFAACAA-OH
Expiry date: May 05, 2020
Supplier: JPT Peptide Technologies GmbH

Lysine peptide
Name: Lysine peptide
Batch no: 211119HS-MHe
Storage: at -20°C or below
Purity: 96.66 %
Molecular weight: 775.91 g/mol
Sequence: Ac-RFAAKAA-OH
Expiry date: April 30, 2020
Supplier: JPT Peptide Technologies GmbH

HPLC System Conditions
HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradienttwice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow

Mobile phases for HPLC:
Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

EVALUATION METHOD OF EXPERIMENTAL DATA
Percent peptide depletion
The concentration of the peptide was determined in each sample and positive control, from absorbance at 220 nm measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the calibration standards.
The percent peptide depletion is determined in each reaction sample and positive control measuring the quotient of the peak area and the mean respective reference control C peak area, according to the formula described below.
peptide percent depletion = [1- { (peak area of the reaction sample or pc) / (mean peak area of reference controls C) } ] × 100

Presence of precipitate
If precipitation occurs it is recorded and caution is used in interpreting data. Samples can be centrifuged to settle and remove the precipitate to avoid clogging the HPLC. Centrifugation at low speed (max. 400 x g) is recorded as well.

Co-elution
In cases where a test chemical co-elutes with the lysine peptide, the Cysteine 1:10-only prediction model can be used. In cases where the test chemical co-elutes with the cysteine peptide and the peptide peak cannot be integrated, a determination of reactivity cannot be made based on the Percent Depletion data from the lysine reaction alone, and the data is reported as “inconclusive”. If the peak for the cysteine peak can be integrated, the instructions below are followed to determine an estimated Percent Depletion.

Negative depletion values
If the Percent Peptide Depletion is < - 10.0%, it is considered that this may be a situation of co-elution, inaccurate peptide addition to the reaction mixture or just baseline “noise.” If this happens, the co-elution controls are carefully analyzed. If the peptide peak appears at the proper retention time and has the appropriate peak shape, the peak can be integrated. The calculated percent peptide depletion is reported as an “estimate“. If this was only an issue for lysine, use the “cysteine-only” prediction model.
If this is an issue with cysteine or both cysteine and lysine, prediction is made based on Table 4. If the peak does not have the proper shape due to complete overlap in retention time of the test chemical and peptide and cannot be integrated, calculation of Percent Peptide Depletion is not possible. If this is an issue for lysine, use the “cysteine-only” model. If this is an issue for cysteine or both cysteine and lysine, the data is reported as “inconclusive”.

Co-elution Controls
If a chemical (Co-elution Control) absorbs at 220 nm and has a similar retention time as a peptide (Reference Control) and the peaks are overlapping, then co-elution of the test chemical with the peptide is reported.
In order to assure that baseline noise is not being called interference it is checked, if the “interfering” chemical peak has a peak area that is >10% of the mean peptide peak area in the appropriate Reference Control. If co-elution occurs, proper integration and calculation of Percent Peptide Depletion is not possible. The data is recorded as “interference” for that peptide.

Co-elution with reactivity and estimated depletion values
In some instances, a test chemical may have an overlapping retention time with either of the peptides and still be reactive with that peptide. This can make the peak area of the peptide appear to be larger than it really is, therefore the calculated percent depletion may be underestimated. If this is the case and the overlap in retention time between the test chemical and peptide is incomplete, percent depletion can still be calculated with a notation of “co-elution – percent depletion estimated”.

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 68.11% ± 1.93% and 69.87 % ± 0.03 while the mean lysine peptide depletion value was 51.03% ± 0.59 %.

Key result

Run / experiment:

other: DPRA Cysteine prediction model, Run 1

Parameter:

other: Mean Depletion Value (%)

Value:

-46.28

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: DPRA Cystein prediciton model, Run 2

Parameter:

other: Mean Depletion Value (%)

Value:

-18.75

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: DPRA Lysine prediction Model

Parameter:

other: Mean Depletion Value (%)

Value:

12.84

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

No runs were rejected. However two runs were conducted with the cysteine peptide in order to make sure that the results of the first run were not due to experimental artifact but were the actual result of action of the test item.

CO-ELUTION
The test chemical absorbs at 220 nm, however the peak area of the co-elution control was not greater than 10 % of the respective reference control C when tested with the cysteine containing peptide (see representative chromatograms in Appendix I), but the retention times were similar. Still, in the reaction samples there were much greater areas integrated than expected, resulting in significantly negative depletion values.
In some instances, a test chemical may have an overlapping retention time with either of the peptides and still be reactive with that peptide. This can make the peak area of the peptide appear to be larger than it really is, therefore the calculated percent depletion may be underestimated.
No co-elution was observed with lysine peptides, though.
The ranges of retention time for cysteine peptide was between 8.420 and 8.530 and between 8.433 and 8.527 while the range of retention time for lysine peptide was between 6.101 and 6.254.

SYSTEM SUITABILITY
Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.51 mM and 0.50 mM for the cysteine and 0.50 mM for the lysine peptide.
A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions from the peptide stock solutions. Calibration standard points were analysed by linear regression.
Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9977 for both cysteine peptides and r2 = 0.9999 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. All validity criteria were within acceptable limits and therefore the study can be considered valid.

ANALYSIS SEQUENCES
Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion.
Moreover the CV % for the nine reference control B and C replicates in acetonitrile (acn) were much smaller than the acceptable 15 % for both peptides, since it was 5.3 % and 1.6 % for cysteine and 0.2 % for lysine peptides. All validity criteria were within acceptable limits and therefore the study can be considered valid.

CYSTEINE AND LYSINE DEPLETION AND MEAN PEPTIDE DEPLETION OF THE TEST ITEM
The mean cysteine peptide concentrations of the reference control C (solvent –acetonitrile) were within the acceptable 0.50 ± 0.05 mM range.
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 68.11% ± 1.93% and 69.87 % ± 0.03 while the mean lysine peptide depletion value was 51.03% ± 0.59 %.
The percent cysteine peptide depletion with the test item was - 46.28% ± 11.92% and
- 18.75 % ± 1.45% for the first and second runs while the percent lysine peptide depletion with the test item was 12.84 % ± 1.09 %. Normally the negative depletion values are substituted with zero when calculating the mean. In this case, although co-elution control area was not greater than 10 % of the reference control C area, cysteine peptide depletion could be underestimated since the values of - 46.28% ± 11.92% and - 18.75 % ± 1.45% are unlikely the result of baseline noise but of an interaction between the test item and cysteine containing synthetic peptide (which could not be observed in the co-elution control alone).

ASSIGNING THE TEST CHEMICAL TO A REACTIVITY CLASS AND CATEGORY
The average percent peptide depletion was calculated for the test item. Since co-elution was observed, applying the cysteine 1:10 / lysine 1:50 prediction model (Table 5) was not possible. When such co-elution and negative depletion values with cysteine peptide happen, discrimination between a skin sensitizer and a non-sensitizer is not possible since cysteine depletion is most likely underestimated moreover there is no only lysine 1:50 prediction model to be applied. The mean percent peptide depletion value was not calculated for the test item, but the study is considered inconclusive.

PROTOCOL DEVIATIONS
Concerning: HPLC analysis time after the ~ 24 h incubation
According to the Study Plan: The total HPLC analysis time should be less than 30 hours.
Modified to: The total HPLC analysis time should be less than 21 hours.
Reason: Change is made based on the acetonitrile batch validation results (Study number: 392-442-5407).
Presumed effect on the study: None

DEMONSTRATION OF TECHNICAL PROFICIENCY
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the inconclusive results with the cysteine peptide run and the fact that there is no applicable “only lysine 1:50 prediction model”, the test item “1,4-Dithiothreitol” (CAS 3483-12-3) could not be assigned to any reactivity class under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method, therefore the study was determined as inconclusive.

Executive summary:

In the course of this study the skin sensitization potential of the test item “1,4-Dithiothreitol” (CAS 3483-12-3) was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction three independent tests were conducted, two with cysteine and one with lysine peptides. Two runs were conducted with the cysteine peptide in order to make sure that the results of the first run were not due to experimental artifact but were the actual result of action of the test item.

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both runs with the cysteine peptide and with the lysine peptide. The cysteine peptide depletion values were 68.11% ± 1.93% and 69.87 % ± 0.03 while the mean lysine peptide depletion value was 51.03% ± 0.59 %.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine runs (0.48 – 0.51 mM and 0.49 – 0.50 mM) and lysine run (0.50 mM) and the CV % for the nine reference controls B and C in acetonitrile were 5.3 %, 1.6 % and 0.2 % percentages for the two cysteine runs and the lysine run respectively. For each peptide, all validity criteria were met, confirming the validity of the study.

The percent cysteine peptide depletion value of the test item was calculated for both runs, however in both cases was reported inconclusive, since the depletion could be underestimated due to co-elution and outstandingly negative depletion values. In some instances, a test chemical may have an overlapping retention time with either of the peptides and still be reactive with that peptide. This can make the peak area of the peptide appear to be larger than it really is, therefore the calculated percent depletion may be underestimated.

The percent lysine peptide depletion could be calculated though and it was 12.84 % ± 1.09 %.

All together, no mean depletion value could be calculated for the peptides, thus the categorization of the test item in one of the four classes of reactivity was not possible.

Based on the inconclusive results with the cysteine peptide run and the fact that there is no applicable “only lysine 1:50 prediction model”, the test item “1,4-Dithiothreitol” could not be assigned to any reactivity class under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method, therefore the study was determined as inconclusive.

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

14 January 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

1. SOFTWARE: DEREX NEXUS

2. MODEL (incl. version number): DEREK NEXUS 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: [C@H]([C@H](CS)O)(CS)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached

5. APPLICABILITY DOMAIN (OECD principle 3)
- Descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predicitons may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compoint does not activate and alert or reasoning tule then Derek makes a negative prediction. The applicability of the negative prediction to the query compoinds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Structural fragment domain: All structure fragments were found in the DEREK database and consequently the structure falls within the applicability domain of DEREK’s skin sensitization end point. The thiol group falls within the structural fragment domain for alert 432.
- Mechanism domain: The structural fragment of the substance falls within the mechanistic domain of the molecules in the training set on which the alert is based.
-Metabolic domain: The structural fragment of the substance falls within the metabolic domain of the molecules in the training set on which the alert is based.



6. ADEQUACY OF THE RESULT
- Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance to QSARs R.6

Version / remarks:

Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 6.0.1.

Principles of method if other than guideline:

- Knowledge Base: Derek KB 2018 1.1
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

GLP compliance:

no

Specific details on test material used for the study:

SMILES: [C@H]([C@H](CS)O)(CS)O

Key result

Parameter:

other: Alert for skin sensitation

Remarks on result:

other: DEREK NEXUS version 6.0.1 did yield a skin sensitization alert for 1,4-Dithiothreitol (CAS 3483-12-3) based on the presence of two thiol groups and predicted 1,4-Dithiothreitol to be sensitizing to the skin (plausible).

Interpretation of results:

other: Substance is predicted to be a skin sensitizer according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

Conclusions:

DEREK NEXUS version 6.0.1 did yield a skin sensitization alert for 1,4-Dithiothreitol (CAS: 3483-12-3) based on the presence of two thiol groups and predicted 1,4-Dithiothreitol to be sensitizing to the skin (plausible).

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of 1,4-Dithiothreitol (CAS: 3483-12-3) with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures.

LHASA has inserted validation comments for the skin sensitization alerts: The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 6.0.1 did yield a skin sensitization alert for 1,4-Dithiothreitol (CAS: 3483-12-3) based on the presence of two thiol groups and predicted 1,4-Dithiothreitol to be sensitizing to the skin (plausible). Therefore, the substance is predicted to be a skin sensitizer according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 6.0.1 did yield a skin sensitization alert for 1,4-Dithiothreitol (CAS 3483-12-3) based on the presence of two thiol groups and predicted 1,4-Dithiothreitol to be sensitizing to the skin (plausible). One proposed mechanism is a nucleophilic attack of the thiol on disulphide bonds in the skin proteins resulting in a new disulphide bridge, i.e. thiol exchange. Another proposed mechanism is that thiol groups can oxidize into free radical generators subsequently reacting with endogenous skin proteins. DEREK NEXUS predicted an EC3 of 3.4% (moderate sensitizer) for 1,4-Dithiothreitol based on LLNA data from ten structurally related analogues (structural similarity of 17% - 83%). 3-Mercaptopropane-1,2-diol, which has an EC3 of 3.6%, has a high similarity (83%) to the query structure, giving confidence in the DEREK NEXUS prediction.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in acetonitrile at 100 mM. There was no evidence of co-elution of the test item with either Cysteine or Lysine peptide. Peptide depletion was calculated as 12.84 % ± 1.09 % in the Lysine Assay, while the percent cysteine depletion values were reported inconclusive for both runs. Based on the inconclusive results with the cysteine peptide run and the fact that there is no applicable “only lysine 1:50 prediction model”, the test item 1,4-Dithiothreitol (CAS 3483-12-3) could not be assigned to any reactivity class under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method, and the study was determined as inconclusive.

1,4-Dithriothreitol is highly reactive and is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. Moreover, it has a very short half-life at biologically relevant pH. It is unclear how fast it will react with the proteins contained on the culture media for the KeratinoSens™ transgenic cell line used in the Keratinosens assay (OECD 442D) or the THP-1 cells used in the hClat assay (OECD 442E). As a result, there might be reduced availability of the test substance in the culture media which could lead to a false negative result. Moreover, the key mechanism leading to the activation of the Keap1-Nrf2-ARE pathway (which the Keratinosens assay is based on) appears to be the electrophilic reaction of stressors with nucleophilic thiols (cysteine sulfhydryl groups) of Keap-1. Therefore, and according to the guidance, caution should be taken as chemicals that do not act as a sensitisers but are nevertheless chemical stressors may lead to false positive results.

Considering the fact that the OECD 442C (first key event of the adverse outcome pathway) was inconclusive, and together with the high uncertainty regarding the applicability of the two in vitro skin sensitization tests for key event 3 and 4 of the adverse outcome pathway (OECD 442D and OECD 224E), further in vitro testing was not considered to be reliable to obtain the required information on the skin sensitization potential of 1,4-Dithriothreitol. Therefore, a Local Lymph Node Assay (LLNA) was performed according to OECD 429.

A valid Local Lymph Node Assay (LLNA) was performed according to OECD 429 and GLP principles. The test item 1,4-Dithiothreitol (CAS 3483-12-3) was formulated in N,N-Dimethylformamide (DMF) at concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v). No mortality or signs of systemic toxicity were observed during the test. No significantly increased lymphoproliferation compared to the relevant control (DMF) was noted for 1,4-Dithiothreitol at the applied test concentrations. The observed stimulation index values were 1.2, 0.8, 0.9 and 1.1 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI = 3) up to the maximum attainable non-toxic, non-irritant concentration of 1 % (w/v) as well as the lack of a significant dose-related response are considered as evidence that 1,4-Dithiothreitol (CAS 3483-12-3) is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DEREK NEXUS version 6.0.1 did yield a skin sensitization alert for 1,4-Dithiothreitol (CAS 3483-12-3) based on the presence of two thiol groups and predicted 1,4-Dithiothreitol to be sensitizing to the skin (plausible). The in vitro test for the first key event of the adverse outcome pathway was inconclusive and due to the chemical properties of the test material, it was concluded that there were uncertainties in the applicability of the in vitro test methods for the second and third key events of the adverse outcome pathway.

As the in vivo LLNA assay was negative and because in vivo studies overrule contradictory results from in silico or in vitro tests, 1,4-Dithiothreitol (CAS 3483-12-3) is not considered to be a skin sensitizer.

In conclusion, the test item is not considered to be a skin sensitizer and does not need be classified for skin sensitization according to Regulation 1272/2008 and amendments.