Butyl carbamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 10, 2016 to August 23, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

These deviations to the study plan, however, did not affect the validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

These deviations to the study plan, however, did not affect the validity of the study

Principles of method if other than guideline:

+ “Chemikaliengesetz” (Chemicals Act) of the Federal Republic of Germany, “Anhang 1” (Annex 1) in its currently valid version

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl carbamate

EC Number:

209-751-0

EC Name:

Butyl carbamate

Cas Number:

592-35-8

Molecular formula:

C5H11NO2

IUPAC Name:

butyl carbamate

Test material form:

solid

Details on test material:

organic

Specific details on test material used for the study:

Batch: #120039110
Purity: >95% Butyl carbamate; <5% n-Butanol
Appearance: White solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Main study: 8 - 9 weeks
Acclimatization: at least 5 days
Temperature: 22 ± 2°C; Relative humidity approx.: 45-65%; Artificial light 6.00 a.m. - 6.00 p.m.
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Concentrations of 10, 25, and 50%. The highest concentration tested was the highest concentration that could technically be achieved.

No. of animals per dose:

4

Details on study design:

Test substance administration:
- Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (`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).

Administration of 3H-methyl-thymidine
- Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
- Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).

Preparation of Single Cell Suspensions
- Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
- The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Clinical Observations:
- All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Ear thickness: In the pre-test, the ear thickness was determined prior to the first application (day 1), on day 3, and prior to sacrifice on day 6.
Ear weights: In the pre-test, the ear weight was determined after sacrifice (biopsy punches were taken from each ear).

- Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weight measures. All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:

Twenty-five percent α-Hexylcinnamaldehyde (vehicle: acetone:olive oil (4+1, v/v)) gave a S.I. of 7.84 and was therefore regarded as a sensitiser in the LLNA test, since the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.28

Test group / Remarks:

test substance concentration: 10%

Remarks on result:

other: No skin sensitisation observed

Results 2

Key result

Parameter:

SI

Value:

0.85

Test group / Remarks:

test substance concentration: 25%

Remarks on result:

other: No skin sensitisation observed

Results 3

Key result

Parameter:

SI

Value:

0.95

Test group / Remarks:

test substance concentration: 50%

Remarks on result:

other: No skin sensitisation observed

Cellular proliferation data / Observations:

- Stimulation Indices (S.I.) of 1.28, 0.85, and 0.95 were determined with the test substance at concentrations of 10, 25, and 50% in DMF, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test substance was not a skin sensitiser under the test conditions of this study.

- Viability / Mortality
No deaths occurred during the study period.

- Clinical Signs
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

- 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.

Any other information on results incl. tables

Interpretation of the results:

The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of ³HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test substance is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I. The estimated concentration of test substance required to produce a S.I. of 3 is referred to as the EC3 value.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Remarks:

not classified

Conclusions:

Under the study conditions, the test substance was not considered to be a skin sensitiser (LLNA test).

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 and EU Method B.42 (LLNA test), in compliance with GLP. Three groups each of four female mice were treated once daily with the test substance at concentrations of 10, 25, and 50% in DMF by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in ab-scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. The body weight was not modified by the treatment. In this study, stimulation indices (S.I.) of 1.28, 0.85, and 0.95 were determined with the test substance at respective concentrations of 10, 25, and 50% in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. Under the study conditions, the test substance was not considered to be a skin sensitiser (Dony, 2016).