Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-24 - 2016-04-20 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Adopted: 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Slovak national Accreditation Service, No. G-026

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

EC Number:

242-644-7

EC Name:

Sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate

Cas Number:

18880-36-9

Molecular formula:

C6H13NO3S3.Na

IUPAC Name:

sodium 3-[(dimethylcarbamothioyl)sulfanyl]propane-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS
- Substance type: pure substance
- Storage condition of test material: The test item was stored in a tightly closed vessel at 15.2- 20.9 °C under dry conditions in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AnLab Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16.84 - 21.23 g
- Housing: The animals were housed individually in plastic cages suspended on stainless steel racks, in a room equipped with central air-conditioning. Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany
- Diet (e.g. ad libitum): A laboratory food Altromin (Altromin Spezialfutter GmbH, Germany) was served ad libitum, each day approximately at the same time.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodical monitored (including microbiological control) and recorded.
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 7 days prior to the start of treatment. The acclimation was according to standard operation procedures.
- Indication of any skin lesions: none noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle
- IN-LIFE DATES: From: 16.03.2016 To: 20.04.2016

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100, 50, 25 %. To achieve 100%, 1g of the test item was mixed with 1ml of the vehicle

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation.
- Compound solubility: The test item was insoluble and a homogeneous suspension was obtained. The required amount of the test item (according to the concentration) was mixed with the appropriate vehicle shortly before the administration.
- Irritation / Systemic toxicity: All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any sign of irritation.
- Ear thickness measurements: Ear thickness wasere measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.
- Erythema scores:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI ≥ 3.

TREATMENT PREPARATION AND ADMINISTRATION:
The required amount of the test item (according to the concentration) was mixed with the appropriate vehicle shortly before the administration. The test substance was administrated in volume of 25 µL to the dorsum of each ear. The alpha-Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test substance, or positive control or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal were recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Five hours later, the animals were killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Positive control substance(s):

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

Statistics:

For calculation of mean and S.D. values of body weights and ear thickness MS Excel was used.

Results and discussion

Positive control results:

Lymph node weight: 0.0663 g, DPM: 6811, SI = 6.23
Results are hence valid

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.79

Test group / Remarks:

100% test item

Results 2

Parameter:

SI

Value:

1.57

Test group / Remarks:

50% test item

Results 3

Parameter:

SI

Value:

1.28

Test group / Remarks:

25% test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph Node Proliferation
In comparison with the control group, an increase of the pooled lymph node weights was recorded in all tested concentrations. This increase was dose dependent. The pooled lymph node weights of treated groups were 0.0260g for 25% concentration, 0.0294g for 50% concentration and 0.0295g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0301g and 0.0663g, respectively. The DPM values for the three treated groups were 1097 (25%), 1345 (50%) and 1540 (100%), respectively. The SI values for the three treated groups were 1.28 (25%), 1.57 (50%) and 1.79 (100%), respectively.

EC3 CALCULATION
The EC3 value could not be calculated, as all measured points were below the stimulation index of three.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

Any other information on results incl. tables

RESULTS

 

Pre-screen test

After the test item application at concentration of 100% signs of local irritation at the application site or systemic toxicity were not observed.

The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the test. The individual and mean values of animal body weights of the treated group are shown here:

 

Table Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weight
1	17.89	20.69
2	19.09	20.08
Mean	18.49	20.38
S.D.	0.848	0.431

 

No erythema was observed in both mice after test item administration (Table below).

 

Table Erythema score

Mouse no.	Day 1	Day 2	Day 3	Day 4	Day 5
1	0	0	0	0	0
2	0	0	0	0	0

 

On day 1(pre dose), day 3 and day 6 the ear thickness was measured. The results are shown in the following Table.

 

Table Ear thickness (mm)

Mouse no.	Day 1 (pre dose)		Day 3		Day 6
1	0.23	0.23	0.24	0.23	0.23	0.24
2	0.25	0.24	0.24	0.24	0.24	0.24
Mean	0.237		0.237		0.237
S.D.	0.009		0.005		0.005

 

The erythema score and the increase in ear thickness did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥25%).

For calculation of mean and S.D. values of body weights and ear thickness MS Excel was used.

 

 

Main Study

 

Clinical Observations

Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity.The daily clinical observation of the animals did not show visible clinical signs.

 

Body Weights

The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

The individual and mean values of animal body weights of the control group, positive control group and the three test item treated groups are shown below.

For calculation of mean and S.D. values of body weights MS Excel was used.

 

Table Individual body weights (g) 

Control (vehicle)
Mouse no.	Initial body weight	Terminal body weight
1	18.32	49.08
2	18.35	19.43
3	18.45	18.69
4	18.59	20.61
5	17.98	19.08
Mean	18.34	19.34
S.D.	0.261	0.739
Positive control
Mouse no.	Initial body weight	Terminal body weight
1	16.84	17.09
2	17.23	18.79
3	18.67	21.04
4	17.85	21.09
5	17.82	20.25
Mean	17.68	19.65
S.D.	0.696	1.707
Sodium 3[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS(100%)
Mouse no.	Initial body weight	Terminal body weight
1	21.23	21.76
2	21.20	22.75
3	21.09	21.17
4	20.81	20.83
5	21.19	21.08
Mean	21.10	21.52
S.D.	0.172	0.768
Sodium 3[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS(50%)
Mouse no.	Initial body weight	Terminal body weight
1	19.09	19.47
2	19.27	21.44
3	18.70	18.97
4	19.08	19.56
5	18.69	19.58
Mean	18.96	19.81
S.D.	0.258	0.942
Sodium 3[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS(25%)
Mouse no.	Initial body weight	Terminal body weight
1	20.66	20.62
2	20.15	21.76
3	19.16	21.11
4	19.94	20.27
5	20.07	21.10
Mean	19.99	21.03
S.D.	0.541	0.532

 

Lymph Node Proliferation

In comparison with the control group, an increase of the pooled lymph node weights was recorded in all tested concentrations. This increase was dose dependent. The pooled lymph node weights of treated groups were 0.0260g for 25% concentration, 0.0294g for 50% concentration and 0.0295g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0301g and 0.0663g, respectively. The DPM values for the three treated groups were 1097 (25%), 1345 (50%) and 1540 (100%), respectively. The SI values for the three treated groups were 1.28 (25%), 1.57 (50%) and 1.79 (100%), respectively. The EC3 value could not be calculated, as all measured points were below the stimulation index of three. The lymph node weights, DPM and SI values are shown here:

 

Table Lymph node weight, DPM, SI, EC3 values.

	Lymph node
	weight (g)	DPM	SI	EC3
Control	0.0301	857	-	-
Positive Control	0.0663	6811	6.23
25%	0.0260	1097	1.28
50%	0.0294	1345	1.57
100%	0.0295	1540	1.79

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate in mice.
All animals survived throughout the test period without showing any clinical signs. In comparison with the control group, the increase in lymph node weight was observed in all treated groups. The increase of lymph node weight was dose dependent. A similar trend was registered in the evaluation of DPM of the lymph nodes. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer. Therefore, it was not possible to calculate an EC3 value.
DPS was identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.

Executive summary:

The sensitization potential of sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS was evaluated using the local lymph node assay (LLNA) according to OECD TG 429 under GLP. The LLNA has been developed to determine the contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline, the test item was formulated in acetone:olive oil 4:1 (v/v) mixture. The positive control (alpha-Hexylcinnamaldehyde) (25%) was formulated in the same vehicle.

The Pre-screen test was performed using dose of 100 % (w/v). Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose for the main test.

Five female mice (CBA/Ca) were topically exposed (dorsum of both ears) to the test solution at concentrations of 100%, 50% and 25%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM values from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25%, 50% and 100%) animals did not show visible clinical symptoms or local irritation or systemic toxicity. The SI values for treated groups were 1.28 (25%), 1.57 (50%), and 1.79 (100%). The EC3 value could not be calculated, as all measured points were below the stimulation index of three.

From the results of this study, sodium 3-[[(dimethylamino)thioxomethyl]thio]propanesulphonate, DPS, is not considered a skin sensitizer in the murine local lymph node assay.