1,2-Benzisothiazole, 3-methyl-, 1,1-dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-21 to 2016-09-27

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

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:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

EC Number:

812-907-6

Cas Number:

34989-82-7

Molecular formula:

C8H7NO2S

IUPAC Name:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF (arrival), Good conventional hygienic level during the test
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 16.6 – 19.7 g
- Housing: Grouped caging (4 animals/cage), Type II. polypropylene/polycarbonate
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 21 days
- Indication of any skin lesions: Animals showed no signs of skin lesion.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 2.5 and 1 %

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
Three groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days.
- Compound solubility: The test substance solubility was tested in several standard vehicles with concentrations up tp 100 %. The best solubility was achieved in DMF and DMSO with a maximum concentration of 5 % (w/v). No or not adequate solubility was observed with the other vehicles at this maximum concentration. As DMF is more preferred than DMSO the test item was formulated in DMF.
- Irritation: No sign of significant irritation (indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement) or any other local effect was observed.
- Systemic toxicity: No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: No increase of ≥ 25 % of ear thickness was observed on any day of measurement.
- Erythema scores: No erythema score ≥ 3 was detected.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals were randomly assigned to control and test groups using a randomization scheme based on body weights.
- Criteria used to consider a positive response: A stimulation index of 3 or greater is an indication of a positive result. However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Animals in the treatment groups were topically treated with 25 μL of the relevant vehicles (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There were no treatments on Days 4, 5 and 6.

Positive control substance(s):

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

Statistics:

DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/group and as DPM/mouse.
The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Significance of the dose-response was evaluated by linear regression using the calculated SI values. All calculations were made by Microsoft Excel Software.

Results and discussion

Positive control results:

The positive control group animals were treated with 25 % HCA solution (formulated in AOO) concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate stimulation compared to the relevant control (AOO). The calculated SI value was 9.3.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

5 %

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

2.5

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

1 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
For the 5 % test substance group the mean corrected DPM count was 2220. For the 2.5 % test substance group the mean corrected DPM count was 1889. For the 1 % test substance group the mean DPM count was 1463.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was calculated.
No significant lymphoproliferative response (SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations.

EC3 CALCULATION
Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group. Loss of body weight ≥ 5 % was observed in the AOO control group only (2/4 animals, 7 or 5 % decrease) but the mean body weight did not decrease significantly hence the effect was considered not significant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance showed no skin sensitisation potential in the LLNA test.

Executive summary:

A study was performed to assess the skin sensitization potential of the test material following dermal exposure on the dorsal surface of the ear in the Local Lymph Node Assay according to OECD Guideline 429. The pooled treatment group approach was used in this test. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated using concentration series. The maximum attainable concentration (based on solubility) was 5 % (w/v) in N,N-Dimethylformamide (DMF) or in Dimethyl sulfoxide (DMSO). As DMF is more preferred than DMSO test item formulations were prepared with DMF. According to results of the DRF, where no adverse effect was observed up to the maximum soluble concentration of 5 % (w/v), the test item was examined in the main test at concentrations of 5 %, 2.5 % or 1 % (w/v). Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore 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 = 9.3) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect 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 the test item at the applied test concentrations. The observed stimulation index values were 1.2, 1.0 and 0.8 at test item concentrations of 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.09, r = 0.99, evaluated by linear regression using the SI values). The lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 5 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test substance is not a skin sensitizer and is not classified for skin sensitisation.