Butyl vinyl ether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 October 2014 - 02 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Butyl vinyl ether

EC Number:

203-860-7

EC Name:

Butyl vinyl ether

Cas Number:

111-34-2

Molecular formula:

C6H12O

IUPAC Name:

1-(ethenyloxy)butane

Details on test material:

- Name of test material (as cited in study report): n-Butyl vinyl ether
- Physical state: Colorless to yellow, clear liquid
- Analytical purity: 99.7 area-%
- Homogenicity: The test item appeared to be homogeneous
- Lot/batch No.: 08571556P
- Expiration date of the lot/batch: March 31, 2015
- Storage condition of test material: at room temperature, under Nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-testj;8 – 10 weeks old; Main study: 9 - 10 weeks
- Housing: group housing (5 animals per group) in Makrolon Type III cages, with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): approx. 45 - 65
- Photoperiod (hrs dark / hrs light):artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25%, 50% ,100%

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be
technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using DMF (upon request by the
sponsor) as vehicle. Vortexing was used to formulate the test item.
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.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily
each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs wererecorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined 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.
At the tested concentrations the animals did not show any signs of systemic toxicity. From day 3 to 6, the animals showed an erythema of the ear skin (score 1). Additionally, the animal treated with 50% test item concentration showed scabby ears on days 4 and 5.
Thus, the test item in the main study was assayed at 25, 50, and 100%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment

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.
Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test
item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was
reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be
implicitly adopted.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and DMF was added. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25, 50 and
100% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 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). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.9 µCi of ³H-methyl thymidine (equivalent to 79.6
µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

DETERMINATION OF INCORPORATED ³HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised 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 per animal (2 nodes per animal). 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. 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 ß-scintillation counter. Similarly, background ³HTdR levels were also
measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell
count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed.
Subsequently, individual cell counts were determined using a cell counter (CASY DT, Schärfe System). The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS:
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm
corresponding to 0.5 cm²). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node
cell count, and for the DPM values (group mean DPM ± standard deviation).
Where appropriate, the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made
statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R`
Decision Tree).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 2.11, 4.53, and 12.71 were determined with the test item at concentrations of 25, 50, and 100% (w/w) in DMF, respectively. A clear dose response was observed. The EC3 value was calculated to be 34.2% (w/w).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and - cell count was observed in the mid and high dose group in comparison to the vehicle control group. Additionally, a statistically significant but not biologically relevant increase in lymph node cell count was observed in the low dose. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice (see Ref. 8) was exceeded in the mid and high dose group (index of 2.1 and 3.3, respectively).

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group:

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (DMF)	1217.0	431.5	1.00
25% n-Butyl vinyl ether	2571.2	602.7	2.11
50% n-Butyl vinyl ether	5509.2*	1005.4	4.53
100% n-Butyl vinyl ether	15472.0*	2458.4	12.71

a)  Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within

a group by the number of animals in that group (5 animals)

*   statistically significant (p<0.05).

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 3	25 (a)	2.11 (b)
Test Group 4	50 (c)	4.53 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 34.2% (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

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

- Clinical Signs: No signs of systemic toxicity or local skin erythema were observed during the study period.

- Body Weights: The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

- Lymph Node Weights and Cell Counts:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase was observed in the mid and high dose group in comparison to the vehicle control group. Additionally, a statistically significant but not biologically relevant increase lymph node cell count was observed in the low dose. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the mid and high dose group (index of 2.1 and 3.3, respectively).

- Ear Weights: The measured ear weight of all animals treated were recorded on test day 6 (after necropsy). A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item n-Butyl vinyl ether was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item n-Butyl vinyl ether was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solutions at different concentrations were prepared in the vehicle DMF.

The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 50% showed scabby ears on day 5 only. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

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

In this study Stimulation Indices (S.I.) of 2.11, 4.53, and 12.71 were determined with the test item at concentrations of 25, 50, and 100% (w/w) in DMF, respectively. A clear dose response was observed. The EC3 value was calculated to be 34.2% (w/w).

A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and - cell count was observed in the mid and high dose group in comparison to the vehicle control group. Additionally, a statistically significant but not biologically relevant increase in lymph node cell count was observed in the low dose. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the mid and high dose group (index of 2.1 and 3.3, respectively).

Thus, the test item n-Butyl vinyl ether was found to be a skin sensitizer and an EC3 value of 34.2 % was derived.