3-methyl-2-butenyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study,

Data source

Reference

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-2-butenyl acetate

EC Number:

214-730-4

EC Name:

3-methyl-2-butenyl acetate

Cas Number:

1191-16-8

Molecular formula:

C7H12O2

IUPAC Name:

3-methylbut-2-en-1-yl acetate

Test material form:

other: liquid

Details on test material:

- Name of test material: Prenyl acetate
- Physical state: clear, colorless liquid
- Analytical purity: 99.6 area%
- Batch No.: 00002977L0
- BASF Substance No.: 10/0362-2
- Homogeneity: test substance appeared to be homogeneous
- Storage: at room temperature
- Expiration Date: August 24, 2013

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Pre-test:
- Strain/quality: CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands

Main study:
- Strain/quality: CBA/CaCrl
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom

ENVIRONMENT
- Temperature: 22 + 2°C
- Relative humidity: 31 – 65% (acclimation period), 45 – 65% (pre-test and main study)
- Artificial light: 6.00 a.m. - 6.00 p.m.
- Air changes: about 10 / hour

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50, 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Body weights, ear weights, ear thickness (using a micrometer) and erythema scoring was assessed in 1 animal/ conc. tested (50%, 100% prenyl acetate)


TREATMENT PREPARATION AND ADMINISTRATION:
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 acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface 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.
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.2 µCi of 3HTdR (equivalent to approximately 80.9 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)

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers. 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:

Stimulation Indices (3H-Methyl Thymidine incorporation) of 0.98, 1.10 and 2.92 at concentrations of 25, 50% and 100%. Indices (mean lymph node weight of test item group related to vehicle group): 1.05, 10.3, 1.57 at concentrations of 25, 50% and 100%. Indices (mean lymph node cell count of test item group related to vehicle group): 1.17, 1.16, 2.01 at concentrations of 25, 50% and 100%.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table

Any other information on results incl. tables

Calculation of Stimulation Incices per Dose Group:

Test item concentration	Group Calculation
	Mean DPM per animal^a)	SD^b)	S.I.
Vehicle (acetone:olive oil (4+1 v/v))	1127.8	321.4	1.00
25 % Prenyl acetate	1110.2	224.3	0.98
50 % Prenyl acetate	1243.4	492.6	1.10
100 % Prenyl acetate	3290.4*	972.9	2.92

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

b)     SD = Standard Deviation

*    statistically significant increase vs. control group (p<0.05)

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. Body weights were within the range commonly recorded for animals of this strain and age.

A statistically significant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group. Furthermore, the cutoff-value for the ear weight index of 1.1 (reported for BALB/c mice for a positive response regarding ear skin irritation) was not exceeded in any dose group.