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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 April - 6 May 2020
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22 July 2010
according to 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)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

In vivo test system

Test animals

CBA/Ca Ola Hsd
Details on test animals and environmental conditions:
- 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.

- 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.

Study design: in vivo (LLNA)

1 %, 0.5 %, 0.25 % and 0.1 % (w/v)
No. of animals per dose:
Details on study design:
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)

- 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 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

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)


- 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.

- 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.

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.

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)

Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
test item concentration of 1 % (w/v)
Test group / Remarks:
test item concentration of 0.5 % (w/v)
Test group / Remarks:
test item concentration of 0.25 % (w/v)
Test group / Remarks:
test item concentration of 0.1 % (w/v)
Cellular proliferation data / Observations:
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.

Normal for all groups

Normal for all groups

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
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.