Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

EC Number:

946-245-5

Molecular formula:

C12H22O

IUPAC Name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

Test material form:

liquid

Details on test material:

According to ECHA communication with reference to Annotation number: SUB-C-2114629169-42-01/F, the substance ID for Koavone has been changed with EC# from 939-627-8 to 946-245-5, and the IUPAC name from Reaction mass of (3R,5R)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (3R,5S)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one to Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at initiation of treatment: about 9 weeks
- Weight at initiation of treatment: 18.4-25.2 g
- Housing: 5/cage during acclimation, 4/cage from allocation shortly before start treatment; in macrolon type III cages with wood shavings (Lignocel) as bedding and shreds of paper (Enviro-dri) as environmental enrichment
- Diet: Rat and Mouse no. 3 breeding diet, RM3 (SDS Special Diets Services, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Allocation: by computer randomization proportionately to body weight

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%):45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light

IN-LIFE DATES:
21-26 September 2011 (from start treatment to end in-life period)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50 and 100% (v/v); no adjustment was made for the purity of the test substance

No. of animals per dose:

4 females/dose

Details on study design:

RANGE FINDING TESTS:
No range-finding test was conducted. Based on the known hazard data of the test material it was expected that dermal application of undiluted test material would not cause systemic toxicity and/or excessive local skin irriation.

MAIN STUDY

RELIABILITY CHECK:
The main study included a positive control group treated with hexyl cinnamic aldehyde diluted (25% v/v) in vehicle (acetone/olive oil 4:1 v/v). The report included a summary of a reliability check conducted in February 2011.

TREATMENT PREPARATION AND ADMINISTRATION:
Test material formulation: shortly before each administration (used within 2 hours). Formulations were shaken until visible homogeneity was obtained.
Administration: vehicle, formulated or undiluted test material, or positive control substance was applied topically on the dorsum of both ears, at a volume of 25 μL per ear, once daily on three consecutive days (study days 0, 1 and 2).
On day 5, all mice received an intravenous injection (tail vein) of 250 μL of phosphate-buffered saline (PBS) containing 20 μCi of [3H]-thymidine. Five hours after this injection, the animals were sacrificed by intraperitoneal injection of sodium pentobarbital.

MEASUREMENTS AND OBSERVATIONS:
- General condition and behaviour: checked daily.
- Body weight: on days -1, 0 (first treatment day) and 5 (day of sacrifice).
- Appearance of lymph nodes and ears at sacrifice.
- 3H-thymidine incorporation in draining auricular lymph nodes (ALN): ALN (pooled per animal) were transfered into PBS and gently rubbed between the rough ends of microscopic slides to suspend the cells. After washing (twice), the cells were resuspended in 5% trichloroacetic acid, left for about 18 hours at 2-10°C, and centrifuged. After removal of the supernatant, the precipitate was left for 24 hours in 1 mL of 1.5 M KOH in 20% EtOH. Next, the solution was transferred into a scintillation vial and 20 mL liquid scintillation cocktail (Hionic Fluor, Perkin Elmer) was added. The vial was thoroughly shaken and the number of disintegrations per minute (DPM) was determined by counting for five minutes in a liquid scintillation counter (Perkin Elmer).

CALCULATION AND EVALUATION OF STIMULATION INDEX (SI):
The SI was calculated by dividing the individual DPM values (corrected for background) by the mean DPM value of the vehicle control group.
The decision process with regard to a positive response included a group mean SI of 3 or higher together with consideration of dose response and statistical analyses based on the test guideline and the recommendations done by ICCVAM.

Positive control substance(s):

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

Statistics:

Body weight and DPM data of the test material groups were compared with the vehicle control group using Dunnett's multiple comparison test. Probability values of <0.05 were considered significant.

Results and discussion

Positive control results:

The SI for the concurrent positive control was 4.3 (mean DPM 5755; p<0.01) and met the criterion for a positive response. This positive response was comparable with that seen in a reliability check with the same positive control substance conducted previously under similar experimental conditions.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

64

Results 2

Key result

Parameter:

SI

Value:

2.31

Test group / Remarks:

25%

Remarks on result:

other: 25%

Results 3

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

50%

Remarks on result:

other: 50%

Results 4

Key result

Parameter:

SI

Value:

3.6

Test group / Remarks:

100%

Remarks on result:

other: 100%

Cellular proliferation data / Observations:

- Clinical signs: no treatment-related abnormalities.
- Body weight: not affected.
- Necropsy findings: no treatment-related findings (unilateral reddish discolouration of an auricular lymph node was seen in 2/4 vehicle controls, 1 mouse of the 25% dose and 1 mouse of the 100% dose).

Any other information on results incl. tables

Since the SI of undiluted test material was >3 and the SI increased with dose, the test material should be regarded as a skin sensitiser when applied undiluted. The EC3 (Estimated Concentration needed to produce a SI of 3) was 64% v/v (calculated from y = 0.017 + 1.91 where y = concentration test material in vehicle and x = SI; R²= 0.9973). 

Applicant's summary and conclusion

Interpretation of results:

other: Substance is a skin sensitiser (1B) in accordance with EU CLP (1272/2008 and its amendments)

Conclusions:

Koavone is sensitising to skin

Executive summary:

In a GLP-compliant study, performed according to OECD Guideline 429 (Local Lymph Node Assay in mice), four groups of four female mice were treated by topical application to the dorsal surface of each ear lobe with 0% (vehicle control), 25%, 50% and 100% solutions of Koavone in acetone/olive oil (4:1 v/v) for three consecutive days. A positive control group of four female mice was treated with hexyl cinnamic aldehyde (25% v/v in the same vehicle). Five days after the first topical application, the mice were injected intravenously (tail vein) with3H-thymidine. Five hours thereafter, the draining auricular lymph nodes were excised, pooled per animal, processed in physiologically-buffered saline, and analysed for lymphocyte proliferation by measuring the incorporation of 3H-thymidine in a scintillation counter. The stimulation index (SI) was 2.31, 2.80 and 3.60 for the 25%, 50% and 100% solution of Koavone, respectively. The SI for the positive control was 4.3 and in agreement with a previous reliability check. Based on the results of this study, Koavone is considered to give a positive result in the LLNA test. An EC3 value of 64% (v/v) was calculated based on the study results.