3-methyltetrahydrothiophene 1,1-dioxide

Endpoint summary

Description of key information

Skin sensitisation (OECDTG422B): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-07-2021 to 30-08-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Version / remarks:

2018-06-25

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Studies of Chemicals, Chapter 5 Health impact test field, Section 35(Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA) Notification No. 2020-46, National Institute of Environmental Research, Republic of Korea

Version / remarks:

2020-11-03

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Specific details on test material used for the study:

Lot no. XXZ2J
Purity : 99.9% (GC)

Species:

mouse

Strain:

CBA

Remarks:

JCrHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: KOATECH_Korea(181-21 Jeonwi-ro, Jinwei-myeon, Pyeongtaek-si, Gyeonggi-do, Republic of Korea, 17711)
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: Specific Pathogen Free(SPF)
- Age at study initiation: 8 weeks (pre-screen and main study)
- Weight at study initiation: 23.1-24.2 g (pre-screen) and 21.4-24.5 (main study)
- Housing: Polycarbonate cage (270 W x 225 L x 130 H mm), less than 5 animals/cage. SAFE® FS-14[73494 Rosenberg(Germany)] bedding was used after sterilizing at 121 ̊C for 20 minutes and the contamination identification was confirmed by receiving a report from the manufacturer.
- Diet: Radiation-sterilized animal feed for rats, 1314 IRR[Altromin Spezialfutter GmbH & Co. KG(Im Seelenkamp 20, D-32791 Lage Postfach 11 20, D-32770 Lage_Germany)] was given ad libitum, and the contamination identification was confirmed by receiving a report from the manufacturer.
- Water: Tap water was filtered and sterilized by ultraviolet sterilizer and microfiltration device and was given ad libitum in 300 mL polycarbonate water bottles. The water was periodically analyzed in accordance with SOP of CentralBio Co., Ltd.
- Acclimation period: Upon receipt, all animals were kept in quarantine and acclimated for 8 days under the environment of the animal room of Non-Clinical Center, CentralBio Co., Ltd. During this period, all animals were observed daily for any clinical signs and only selected healthy animals will be used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6~23.0 ºC(Pre-screen test I), 20.4~23.1 ºC(Pre-screen test II), 20.7~23.4 ºC(Main test)
- Humidity (%): 53.8~64.6 %(Pre-screen test I), 50.3~64.4 %(Pre-screen test II), 49.2~58.3 %(Main test)
- Air changes (per hr): 10~15/hr.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark (On: 8 AM, Off: 8 PM, controlled by automated timer), 150~300 lux
- IN-LIFE DATES: From: 20 July 2021 To: 30 August 2021

Vehicle:

dimethyl sulphoxide

Remarks:

The test substance was dissolved in DMSO as a result of a previous vehicle test. DMSO was confirmed to be an appropriate vehicle as it would ease the test substance administration and have no effect on the test system(animal).

Concentration:

During the pre-screen test, the concentrations of 100 %, 50 %(w/v), 25 %(w/v), 10 %(w/v) and 5 %(w/v) were selected. The skin response was evaluated as "0" for all animals. No animals were observed to have lost body weight by more than 5 % when compared body weight on the treatment day to day 6 and no animals were observed to have increased ear thickness by more than 25 % when compared measurement on the treatment day to day 3 or day 6. Furthermore, no dead or moribund animals were observed. Therefore, 100 % was selected as the highest concentration in accordance with the pre-screen test result and “Testing Guidelines for Studies of Chemicals". Then 50 %(w/v), 25 %(w/v) were selected as the mid and lowest concentrations.

No. of animals per dose:

Pre-screen: 1
Main study: 4

Details on study design:

PRE-SCREEN TESTS: See Table 1 in Any other information
- Compound solubility: not described
- Mortality: All animals were observed daily
- Clinical signs / systemic toxicity: All animals were observed daily for clinical signs.
- Local skin irritations: All animals were observed daily for clinical signs.
- Body weight: measured on the day of treatment (Day 1/immediately prior to treatment) and at the end of the test (Day 6)
- Ear thickness measurements: The ear thickness of the left and right ear of each mouse were measured on Day 1(before treatment), Day 3, and Day 6 to verify whether the thickness of the ear on Day 3 or Day 6 has increased by more than 25 % compared to Day 1. In addition, the ear thickness of the test substance group was checked for statistically significant change compared to negative control group.
- Erythema scores: For all animals during the pre-screen test and the main test, the treatment sites were observed once daily for the local skin reactions and the scores were recorded in accordance with "[Table 2 in Any other information] Erythema Scores". The result of the pre-screen test was used to set the concentration of the main test, and the results of the main test was used to assess the skin sensitization potential of the test substance.

MAIN STUDY: See Table 3 in Any other information
Same as PRE-SCREEN TEST and additionally the Stimulation Index (SI):
The results of each test group were evaluated by the Stimulation Index(SI). The calculation of the stimuli index was derived according to the "Stimulation Index Calculation Formula" below (see Any other information). Using the calculated evaluation values, the SI value of the Acetone:Olive oil (4:1, v/v) group and DMSO group were determined to be 1.0, and the positive control group were calculated by comparing the Acetone:Olive oil(4:1, v/v) group. The test substance group was calculated by comparing the DMSO group. The test was judged to be reliable as the absorbance value of the negative control group was between 0.1 and 0.2. It was also judged to be positive and reliable as the SI of the positive control group did exceed 1.6 but not 14. The decision process regards a result as positive when SI ≥ 1.6

ANIMAL ASSIGNMENT AND TREATMENT
Following the quarantine-acclimation period, in both pre-screen test and main test, selected healthy animals were randomly grouped based on body weights. The evenness for the average and standard deviation of body weights per group was checked during the group assignment.
Day 1~Day 3: Treatment of substance:
The test substance, vehicle, and positive control were treated over the entire dorsal surface of the ear once a day up to Day 3 using a micropipette at 25 μL/ear to each corresponding group.
Day 4: No Treatment
Day 5: BrdU solution injection:
Intraperitoneal (IP) injection 0.5 mL(5 mg/mouse) of BrdU at a concentration of 10 mg/mL solution approximately 24 hours prior to the extraction of the auricular lymph node.
Day 6: Auricular lymph node extraction:
Approximately 24 hours after BrdU injection, all animals were euthanized with CO2 gas. The auricular lymph nodes were excised from each mouse and gently disassembled using 70 micro-mesh(SPL, Cell Strainer) and distributed with PBS(1×) for a total volume of 15 mL.
Day 6: Cellular proliferation measurement:
BrdU intensity was measured by ELISA using a commercial kit. Briefly, 100 μL of the lymph node cells(LNC) suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and then the substrate solution was added and allowed to produce chromogen. Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured.
TREATMENT PREPARATION AND ADMINISTRATION:
Pre-screen: Test substance was applied without any correction for purity. The required amount of the test substance was weighed by an electronic balance and placed in a tube. A small amount of vehicle was added and mixed using a vortex mixer until dissolved. In the pre-screen test, the test substance was prepared at concentrations of 5 %(w/v), 10 %(w/v), 25 %(w/v) 50 %(w/v) and 100 %.
Main study: In the same way as the preliminary test, using DMSO, a test substance adjuvant, the test substance was prepared at concentrations of 25 %(w/v), 50 %(w/v) and 100 %. All preparations were performed on the day of treatment.

Positive control substance(s):

eugenol (CAS No 97-53-0)

Statistics:

The SPSS Statistical Program (IBM, Ver. 25) was used to conduct statistical analysis of the body weights and the ear thickness. After testing the normality of the variance (Anderson Darling or other appropriate method), statistics was processed as One-way ANOVA test. Then, homogeneity of variances was verified with Levene’s test. Confirmed as a case of homogeneity, the Dunnett test was conducted as a post-hoc test to confirm the significance with the control group. P-values<0.05 was considered statistically significant.

Positive control results:

The Positive control Eugenol did produce a positive LLNA with an SI of 2.823. It therefor is judged to be positive and reliable as the SI did exceed 1.6 but did not exceed 14.

Key result

Parameter:

SI

Value:

0.773

Remarks on result:

other: 25%

Key result

Parameter:

SI

Value:

0.865

Remarks on result:

other: 50%

Key result

Parameter:

SI

Value:

0.897

Remarks on result:

other: 100%

Table 4: Stimulation Index (main test)

Groep	Concentration	Sex		Absorbance		BrdU index	SI index
				370 nm	492 nm
Blank	-	-	Mean	0.083	0.040	-	-
			S.D.	0.002	0.000	-
			N^a	3	3	-
G1	Acetone:Olive oil 4:1 (v/v)	Female	Mean	0.229	0.045	0.141	1.000
			S.D.	0.061	0.003	0.059
			N	4	44	4
G2	DMSO	Female	Mean	0.276	0.048	0.185	1.000
			S.D.	0.043	0.001	0.041
			N	4	4	4
G3	Eugenol 25%(w/v)	Female	Mean	0.499	0.058	0.398	2.823
			S.D.	0.112	0.005	0.107
			N	4	4	4
G4	Test substance 25%(w/v)	Female	Mean	0.232	0.046	0.143	0.773
			S.D.	0.040	0.002	0.038
			N	4	4	4
G5	Test substance 50%(w/v)	Female	Mean	0.250	0.047	0.160	0.865
			S.D.	0.038	0.002	0.036
			N	4	4	4
G6	Test substance 100%(w/v)	Female	Mean	0.256	0.047	0.166	0.897
			S.D.	0.059	0.002	0.056
			N	4	4	4

-: Not applicable

a: Number of total wells

N: Number of animals

S.D. Standard deviation

Test substance: 3-methylsulfolane

 

PRE-SCREEN TESTS: 

- Mortality: not observed

- Clinical signs / systemic toxicity: not observed

- Local skin irritations: not observed

- Body weight: no body weight loss > 5% observed

- Ear thickness measurements: no increase > 25% observed

- Erythema scores: not observed

 

MAIN STUDY

- Mortality: not observed

- Clinical signs / systemic toxicity: not observed

- Local skin irritation: not observed

- Body weight: no significant body weight change or body weight loss > 5% observed

- Ear thickness measurements: no significant change in ear thickness or increase > 25% observed

- Erythema scores: not observed

 

Interpretation of results:

other: Substance is not a skin sensitiser (1A or 1B) - based on CLP criteria (Annex I 1272/2008/EC)

Conclusions:

The substance is not a skin sensitiser in the Local Lymph Node Assay : BrdU-ELISA (OECD guideline 422B)
Based on these results the test substance does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

This study was conducted to assess the skin sensitisation potential of the test substance by measuring the proliferation of lymphocytes in the auricular lymph nodes through 5-Bromo-2'-Deoxyuridine content analysis following applications to female CBA/J mice (OECD 422B).

In order to set the dose concentration of the main test, the pre-screen test was conducted with concentrations of 5 % (w/v), 10 % (w/v), 25 % (w/v), 50 % (w/v) and 100 % using 1 animal per concentration, and no systemic toxicity and excessive local stimulation were observed.

Based on these results, the concentration of administration in the main test was set at 25% (w/v), 50% (w/v), and 100 % using 4 animals per concentration.

The results of main test are as follows.

- No mortality was observed in all groups.

- No clinical signs were observed in all groups.

- No statistically significant changes in body weights were observed in all treatment groups compared to negative control group and no animals were observed to have lost body weight by more than 5 % when compared body weights on the treatment day to day 6.

- No statistically significant changes in ear thickness were observed in all treatment groups compared to negative control group and no animals were observed to have increased ear thickness by more than 25 % when compared measurements on the treatment day to day 3 or day 6.

- No local skin irritations were observed in all groups.

After administering the test substance at concentrations of 25 % (w/v), 50 % (w/v) and 100 % the Stimulation index (SI) was calculated by measuring the cell proliferation in each lymph node were determined as 0.773, 0.865 and 0.897, respectively. The SI of the positive control, Eugenol 25.0 %(w/v), was calculated as 2.823.

The above results show that the test substance does not cause skin sensitisation at 25 % (w/v), 50 % (w/v) and 100 % concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For 3-methylsulfolane a Local Lymph Node Assay: BrdU-ELISA is performed.

This study was conducted to assess the skin sensitisation potential of the test substance by measuring the proliferation of lymphocytes in the auricular lymph nodes through 5-Bromo-2'-Deoxyuridine content analysis following applications to female CBA/J mice (OECD 422B).

In order to set the dose concentration of the main test, the pre-screen test was conducted with concentrations of 5 % (w/v), 10 % (w/v), 25 % (w/v), 50 % (w/v) and 100 % using 1 animal per concentration, and no systemic toxicity and excessive local stimulation were observed.

Based on these results, the concentration of administration in the main test was set at 25% (w/v), 50% (w/v), and 100 % using 4 animals per concentration.

The results of main test are as follows.

- No mortality was observed in all groups.

- No clinical signs were observed in all groups.

- No statistically significant changes in body weights were observed in all treatment groups compared to negative control group and no animals were observed to have lost body weight by more than 5 % when compared body weights on the treatment day to day 6.

- No statistically significant changes in ear thickness were observed in all treatment groups compared to negative control group and no animals were observed to have increased ear thickness by more than 25 % when compared measurements on the treatment day to day 3 or day 6.

- No local skin irritations were observed in all groups.

After administering the test substance at concentrations of 25 % (w/v), 50 % (w/v) and 100 % the Stimulation index (SI) was calculated by measuring the cell proliferation in each lymph node were determined as 0.773, 0.865 and 0.897, respectively. The SI of the positive control, Eugenol 25.0 %(w/v), was calculated as 2.823.

The above results show that the test substance does not cause skin sensitisation at 25 % (w/v), 50 % (w/v) and 100 % concentrations.

Justification for classification or non-classification

Based on the available information, 3-methyl sulfolane does not need to be classified for skin sensitisation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).