5H-1,2-oxathiole 2,2-dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.09.2019 - 02.12.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5H-1,2λ⁶-oxathiole-2,2-dione

EC Number:

606-834-7

Cas Number:

21806-61-1

Molecular formula:

C3H4O3S

IUPAC Name:

5H-1,2λ⁶-oxathiole-2,2-dione

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Shijiazhuang Suntec-chem Co., Ltd.; 190403
- Expiration date of the lot/batch: Apr 10, 2020
- Purity: 99.92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully resealed.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known: Microbiologically defined background according to internal SOP
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: In pilot experiment: 16.67-17.54 g; In main test: 16.10 - 19.63 g
- Housing: Animals in groups in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany) ad libitum
- Water: Drinking tap water ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: During clinical observations the examination of skin irritation at application site was carried out.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433

Remarks:

Mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol. This vehicle (1) is used in our laboratory for approximately 10 years and it elicited a consistent response over whole period. (1) Ehling et al.. (2005), Toxicol., 212, 60-68.

Concentration:

50%, 5.0%, 0.5% w/v in pilot experiment.
20%, 2.0%, 0.2% w/v in main study.

No. of animals per dose:

1 female per group (pilot)
5 females per group (main test)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Three concentrations were used in pilot experiment: 50%, 5%, 0.5% concentrations in the form of suspension in DAE 433, one mouse per concentration.
- Irritation: The ear weights in pilot experiment were relatively balanced.
- Systemic toxicity: Reduction of body weight after treatment was recorded in all animals during the pilot experiment, however, the weight fluctuations are common in mice.
- Ear thickness measurements: The thickness of ears in all animals during pilot experiment was slightly increased.
- Erythema scores: In treated animals, no erythema and skin reaction were observed.
During pathological examination, the auricular lymph nodes enlargement was not detected in all animals.

According to the specification of the study monitor, the following doses (20%, 2%, 0.2%) were determined for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The response towards the test item is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship).

TREATMENT PREPARATION AND ADMINISTRATION:
In the application period, the test item was administered in the form of a suspension in DAE 433. The volume of the application form was constant at all groups of animals - 25 μL of the appropriate suspension to the dorsum of each ear every morning for 3 consecutive days. The application was performed very slowly by micropipette. The application forms of the test item (suspensions) were prepared immediately before administration.
Day 4 and day 5 were no treatment.
In the day 6, the weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.43 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed and draining auricular lymph nodes were collected. btained lymph nodes were processed with PBS for each treatment group.
Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM).

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB, CAS No 97-00-7)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed by applying the parametric test for testing whether all group samples originate from the same distribution and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Results and discussion

Positive control results:

The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (24.70) and ear weight.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.09

Test group / Remarks:

0.2%

Results 2

Parameter:

SI

Value:

2.06

Test group / Remarks:

2%

Results 3

Parameter:

SI

Value:

3.12

Test group / Remarks:

20%

Results 4

Parameter:

EC3

Value:

17.96

Test group / Remarks:

EC3 = [(3-d)/(b-d)] x (a-c) + c; a=20, b=3.12, c=2, d=2.06

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The value of the DPM and SI for the positive control group (DNCB) was statistically significantly increased. The SI was ≥ 3 (24.70) – the LLNA was efficient (see Table 9).
The SI for the 2% (2.06) and 0.2% (1.09) test item groups was below the threshold, and the stimulation index (SI) is < 3. The SI for the 20% test item group is above the threshold (3.12), and the stimulation index (SI) is ≥3. The value of the DPM at dose levels 20% and 2% was statistically significantly increased compared to the negative control.

DETAILS ON STIMULATION INDEX CALCULATION: Mean values and standard deviations of incorporation of 3H-methyl thymidine were computed for the test item groups and for the positive as well as the vehicle control group. Stimulation Index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

EC3 CALCULATION: The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation: EC3 = [(3-d)/(b-d)] x (a-c) + c.

For the test item, a=20, b=3.12, c=2, d=2.06. Therefore the EC3 value = [(3-2.06)/(3.12-2.06)] x (20-2) + 2 = 17.96%

CLINICAL OBSERVATIONS: During the pilot experiment no clinical symptoms of systemic toxicity were observed. In treated animals, no erythema and skin reaction were observed.
The thickness of ears in all animals during pilot experiment was slightly increased. During pathological examination, the auricular lymph nodes enlargement was not detected in all animals.
No animals died during the main study. All animals from the 20% concentration group of the test item showed hyperemia of the auricle from the second day. No symptoms of toxicity and no erythema on the application site were observed in all animals from the 2% and 0.2% test item groups. All animals in the positive control group showed symptoms caused by the application of DNCB: hyperemia of auricle, clonic spasm and increased response to stimuli (Table 8).

BODY WEIGHTS: Pilot study - Individual body weight of animals before administration was similar. Reduction of body weight after treatment was recorded in all animals during the pilot experiment, however, the weight fluctuations are common in mice (Table 3). Main study - The body weights of treated groups and the positive control group were slightly decreased compared to the negative control animals during the whole study. Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increments were negative at all dose levels (Table 6, 7).

Any other information on results incl. tables

Ear Weights/Redness

In the main test, the ear weight of the positive control was statistically significantly increased compared to the negative control group (Table 10). Ear weight for the 20% test item group was statistically significantly increased compared to the negative control group. No statistically significantly changes of ear weight were recorded in the 2% and 0.2% test item groups.

No erythema of the skin was observed during the clinical observation at 2% and 0.2% dose levels (Table 11). Very slight erythema was observed during the clinical observations at the 20% dose level. Very slight erythema and well-defined erythema in animals of the positive control group were recorded during the study (see Table 11).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Category 1B based on CLP criteria

Conclusions:

In an in vivo LLNA test in Balb/c female mice, 1,3-Propenesultone, demonstrated a positive sensitising response in the LLNA assay. The SI at the 20% dose level was ≥ 3.

Executive summary:

In a dermal sensitization study (152/19/6) with 0, 0.2, 2 and 20% w/v 1,3-Propenesultone (99.92%) in DAE 433 (40 % dimethylacetamide, 30 % acetone and 30 % ethanol), young adult female BALB/c mice (5/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the concurrent testing of the positive control, Dinitrochlorobenzene (DNCB).

The pilot study with 0.5, 5 and 50% w/v 1,3-Propenesultone in DAE433 established the doses for the main study. The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (SI; 24.70) and ear weight.

The test item 1,3-Propenesultone, caused an increase in ear weight at the 20% dose level only, compared to the negative control. The animals exposed to 20% 1,3-Propenesultone showed very slight erythema and hyperemia of auricles during the clinical observations. The animals exposed to 2% and 0.2% 1,3 -Propenesultone showed no skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The DPM values were statistically significantly increased in animals dosed at 20% and 2% 1,3 - Propenesultone compared to the negative control. The value of the SI for the group treated at the 20% dose level was over the threshold of ≥3 (3.12). The value of the SI for groups treated at the 2% and 0.2% dose levels was below the threshold (2.06 and 1.09, respectively). The EC3 was calculated to be 17.96%. Under the given test conditions, the test item, 1,3 - Propenesultone, demonstrated a positive sensitising response in the LLNA assay.