4-methylbenzothiazol-2-ylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated 06 July 2012

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

The relative humidity in the animal room was between approximately 29-65% instead of 45-65% and the temperature was between 20-27°C instead of 20-24°C for several hours. The age of the animals in the first irritation pre-test was 12 – 13 weeks at the start of treatment instead of 8 – 12 weeks. These deviations, however, did not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

4-methylbenzothiazol-2-ylamine

EC Number:

216-028-3

EC Name:

4-methylbenzothiazol-2-ylamine

Cas Number:

1477-42-5

Molecular formula:

C8H8N2S

IUPAC Name:

4-methyl-1,3-benzothiazol-2-amine

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 1st pre-test: 12 - 13 weeks; 2nd pre-test and main study: 10 - 11 weeks
- Weight at study initiation: 1st pre-test: 21.1-23 g; 2nd pre-test: 19.4 - 20.8 g; main test: 17.4 - 21.7 g
- Housing: group; Makrolon Type II (pre-test) / III (main study), with wire mesh top; granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (except for deviations)
- Humidity (%): 45-65% (except for deviations)
- Photoperiod (hrs dark / hrs light): artificial light 6.00 am - 6.00 pm

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

The highest test item concentration, which could be technically used was a 25% solution in DMSO
1st pre-test: 10 and 25%
2nd pre-test: 2.5 and 5%
Main study: 0.5, 1, and 2%

No. of animals per dose:

The highest test item concentration, which could be technically used was a 25% solution in DMSO
1st pre-test: 1
2nd pre-test: 1
Main study: 4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: yes; a solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% solution in DMSO.
- Irritation: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily.
- Systemic toxicity: Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily.
- Ear thickness measurements: In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
- Erythema scores: as set out in OECD 429

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:

Test Item Preparation and Analysis
The test item was placed into an appropriate container on a tared balance and DMSO was added. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.5, 1, and 2% in DMSO. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

3H-Methyl-Thymidine Administration
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.2 μCi of 3H-methyl thymidine (equivalent to 80.7 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

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

Results and discussion

Positive control results:

α-Hexylcinnamaldehyde [vehicle: acetone:olive oil (4+1, v/v)], Stimulation Index = 3.43 for 10% test item group (1.00 for control group); EC3 = 8.7%
Substance was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.85

Test group / Remarks:

0.5% in DMSO

Remarks on result:

other: negative

Results 2

Parameter:

SI

Value:

1.11

Test group / Remarks:

1% in DMSO

Remarks on result:

other: negative

Results 3

Key result

Parameter:

SI

Value:

0.95

Test group / Remarks:

2% in DMSO

Remarks on result:

other: negative

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
In this study Stimulation Indices of 0.85, 1.11, and 0.95 were determined with the test item at concentrations of 0.5, 1, and 2% in DMSO.

EC3 CALCULATION
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were observed during the main study period. From day 1 to 5, animals showed an erythema of the ear skin (Score 1 to 2), but that did not exceed the maximum acceptable limit for skin irritation.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

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. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitization potential of the substance. Under the experimental conditions reported, the test item did not induce a stimulation index over 3 in the test concentrations suitable for dosing and therefore, it is considered not to be a skin sensitiser under the test conditions of this study.

Executive summary:

In the OECD 429 study (GLP) the test item formulated in DMSO (dimethylsulfoxide) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 1, and 2%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments, and as was proposed by the Sponsor and agreed by the Study Director.

All treated animals of the main experiment survived the scheduled study period and no signs of systemic toxicity were observed. From day 1 to 5, animals treated with 0.5, 1.0 or 2.0% of the substance showed a dose-related erythema of the ear skin (Score 1 to 2), but that did not exceed the maximum acceptable limit for skin irritation.

In this study Stimulation Indices (S.I.) of 0.85, 1.11, and 0.95 were determined with the test item at concentrations of 0.5, 1, and 2% in DMSO, respectively. Hence, the test item was not a skin sensitiser under the test conditions of this study.