Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2013 - 22 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

EC Number:

691-061-8

Cas Number:

1001320-38-2

Molecular formula:

C10H19NO2S2

IUPAC Name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

Test material form:

solid

Remarks:

yellow grease-like solid

Specific details on test material used for the study:

Molecular formula: C10H19NO2S2
Name: n-butoxycarbonyl n-butyl dithiocarbamate (active ingredient)
CAS Number: 1001320-38-2

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeld makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 0.5, 1 and 2.5%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Initially, two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids). Concentrations were mixed by shaking or stirring with a vortex mixer or magnetic stirrer immediately prior to dosing.
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days.
Based on the results of the animals treated initially, additional animals were treated in a similar manner with four lower concentrations (1%, 2.5%, 5% and 10%) at a later stage.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. The test substance was melted for a maximum of 3.5 hours at a maximum of 27ºC. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing) according to the OECD numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: no necropsy was conducted on the animals.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.4

Test group / Remarks:

0.5%

Results 2

Parameter:

SI

Value:

2.6

Test group / Remarks:

1%

Results 3

Parameter:

SI

Value:

13.5

Test group / Remarks:

2.5%

Results 4

Parameter:

EC3

Value:

1.1

Any other information on results incl. tables

Results Pre-screen test:

Animals at 25 and 50% showed systemic toxicity with clinical signs including hunched posture, piloerection, deep breathing and ptosis, cold to the touch and/or reduced activity. These animals were only dosed on Days 1 and 2 and were euthanized on Day 3.

Animals at 1 and 2.5% concentrations no signs of systemic toxicity were noted and no irritation was observed, except for one animal at 2.5% who had slight irritation on Day 4. Animals at 5 and 10% had signs of irritation including redness and three of the four animals had swelling of the head on Days 5 and 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was 2.5%.

Other results - main study:

Mean DPM/animal values for the experimental groups treated with test substance concentrations of 0.5, 1 and 2.5% were 438, 788, and 4086 DPM respectively. The mean DPM/animal value for the vehicle control group was 304 DPM.

No irritation of the ears was observed in any of the animals examined.

 

All auricular lymph nodes were considered normal in size except for two animals at 2.5% who had slightly larger nodes (<25% is acceptable increase). No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose, groups was not considered toxicologically relevant since the changes were slight in nature and no concentration-related incidence was apparent.

    

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 2.5%.

Executive summary:

The test material was assessed for contact hypersensitivity in an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, up to 2.5% at which no systemic toxicity was observed.

No irritation of the ears was observed in any of the animals examined. All auricular lymph nodes were considered normal in size except for two animals at 2.5% who had slightly larger nodes, which is acceptable.

The SI values calculated for the substance concentrations 0.5, 1 and 2.5% were 1.4, 2.6 and 13.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 1.1% was calculated.

Based on the results, the test material should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction, according to Regulation (EC) No 1272/2008.