Amines, C12-14-tert-alkyl, compds. with 2(3H)-benzothiazolethione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2018 to 19 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies, 12 NouSan No. 8147, 24 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Amines, C12-14-tert-alkyl, compds. with 2(3H)-benzothiazolethione

EC Number:

272-782-3

EC Name:

Amines, C12-14-tert-alkyl, compds. with 2(3H)-benzothiazolethione

Cas Number:

68911-68-2

Molecular formula:

N/A

IUPAC Name:

C12-14-tert-alkylamines, compds. with 2(3H)-benzothiazolethione

Test material form:

liquid

Specific details on test material used for the study:

- CAS Number: 68911-68-2
- Purity: 99% (nominal); a substance of Unknown or Variable composition, Complex reaction products of biological materials (UVCB)
- Description: Dark Amber viscous liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

The animals were housed in suspended solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flakes bedding (Datesand Ltd., Cheshire, UK).

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 10, 25 and 50 % w/w

No. of animals per dose:

One mouse for the preliminary test, followed by five animals per dose in the main test.

Details on study design:

Preliminary Screening Test
A preliminary screening test was performed using one mouse, treated by daily application of 25 µL of the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed for local skin irritation, ear thickness, any clinical signs of toxicity and body weight.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation and that 50% was the highest suitable concentration, based upon the observation of slight yellow-coloured residual test item on the ears on the mice at that concentration.
The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
- Clinical Observations: All animals were observed twice daily (pre- and post-dose) on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6 for any signs of toxicity or signs of ill health.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation. The lymph node cells were rinsed with PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube and was pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in PBS and re-pelleted. The pellet was re-suspended in 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 ºC, the TCA precipitates were recovered by centrifugation and re-suspended in TCA and transferred to scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting.

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item was regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation was classified as a "non sensitizer".

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

8.65

Test group / Remarks:

10% w/w in acetone/olive oil 4:1

Results 2

Key result

Parameter:

SI

Value:

14.52

Test group / Remarks:

25% w/w in acetone/olive oil 4:1

Results 3

Key result

Parameter:

SI

Value:

18.85

Test group / Remarks:

50% w/w in acetone/olive oil 4:1

Results 4

Key result

Parameter:

EC3

Value:

4.1

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information, the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

 

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
10	8.65	Positive
25	14.52	Positive
50	18.85	Positive

 

Clinical Observations

All animals survived to the schedule euthanasia; there were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Body Weight

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

 

EC3 Value

The concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation (EC3 value) was calculated to be 4.1% (extrapolated).

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the conditions of the test, the test item is classified as a contact sensitizer (Category 1B) according to GHS, with an EC3 value of 4.1%.

Executive summary:

A skin sensitisation study, local lymph node assay in the mouse (individual method) was performed in accordance to the standardized guidelines OECD 429 and EU Method B.42, under GLP conditions in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in a female mouse treated with test item at a concentration of 50% v/v in acetone/olive oil 4:1, in which no clinical signs of toxicity were noted, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five female animals, were treated with 50 μL (25 μL per ear) of the test item, as a solution in acetone/olive oil 4:1, at concentrations of 50%, 25% or 10% v/v. A further group of five female animals was treated with acetone/olive oil 4:1 alone in the same manner.

 

Under the conditions of the test the test item is classified as a contact sensitizer (Category 1B) according to GHS, with an EC3 value of 4.1%.