Acetic acid, anhydride, reaction products with 1,5,10-trimethyl-1,5,9-cyclododecatriene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2007 - 13 June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

-

EC Number:

482-330-9

EC Name:

-

Cas Number:

144020-22-4

Molecular formula:

C17H26O

IUPAC Name:

1-[(1E,5Z,9Z)-2,5,10-trimethylcyclododeca-1,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one; 1-[(1S)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1S)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Bicester, Oxon, England.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: eight to ten weeks of age
- Weight at study initiation: 18.3 to 22.1 g
- Housing: Housed individually in polycarbonate cages with woodflake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

concentrations of 10, 25 and 50% v/v in Acetone : Olive Oil (4:1 v/v)

No. of animals per dose:

four

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method: Local Lymph Node Assay (LLNA) - individual animal approach
- Criteria used to consider a positive response (DSD/CLP): Stimulation Index (SI) > 3
- Selection of dosage levels: The maximum practical concentration for pinna dosing was test 50% v/v. Based on this information the following concentrations were selected for the study: 10, 25 and 50% v/v

TREATMENT PREPARATION AND ADMINISTRATION:

Administration of the test substance
- Groups of four mice were treated by daily application of 25µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days.
- The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.
- The test item was formulated freshly on each day of dosing.

Administration of 3 H-methyl Thymidine
- Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline containing 3 H-methyl Thymidine† (3
HTdR: 80 µCi/ml) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle (26 gauge) after the mouse had been heated in a warming chamber.

OBSERVATIONS
- Clinical signs: All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.
- Bodyweight: The weight of each mouse was recorded on arrival (these data are not reported), Day 1 (first day of dosing) and prior to termination (Day 6).

TERMINAL STUDIES
- Termination: Five hours following the administration of 3 HTdR on Day 6 all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised for each experimental animal. 1.0 ml of phosphate buffered saline was added to the lymph nodes for each animal. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

- Preparation of single cell suspensions: A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding 10 ml phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 ml trichloroacetic acid (TCA: 5%) following the final wash.

- Determination of incorporated 3 H-methyl Thymidine: After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 ml 5% TCA and transferred to 10 ml Ultima gold scintillation fluid on Day 7. HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

Positive control substance(s):

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

Results and discussion

Positive control results:

Positive control study with hexyl cinnamic aldehyde using the local lymph node assay (Study No. HLS/0521). This study was performed to confirm the sensitivity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study was performed using the murine local lymph node assay and a known moderate sensitizer - hexyl cinnamic aldehyde (HCA) [Technical grade 90.6%] supplied by Aldrich Chemical Co, England (Batch No. 13102MO, Expiry 13 June 2007).

This positive control study was conducted between 24 May and 6 June 2007 using sixteen mice of the CBA/Ca strain supplied by Harlan UK Ltd., Bicester, Oxon, England. Groups of four mice were used in this study. Three dosage levels and a vehicle control were used with dosages of HCA selected based on previous experience with HCA at this laboratory as follows: 10, 25 and 50% v/v in AOO (4:1 v/v acetone:olive oil)
The positive control study is considered to be valid if at least one concentration of HCA results in a threefold greater increase in 3H-methyl thymidine (3HTdR) incorporation compared to control values.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

20.71

Results 2

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10% v/v

Remarks on result:

other: 10%

Results 3

Key result

Parameter:

SI

Value:

3.6

Test group / Remarks:

25% v/v

Remarks on result:

other: 25%

Results 4

Key result

Parameter:

SI

Value:

6.8

Test group / Remarks:

50% v/v

Remarks on result:

other: 50%

Any other information on results incl. tables

Mortality and clinical signs: There were no deaths and no signs of ill health, toxicity or irritation observed during this study.

Greasy fur was noted for all control and test animals post-dose from Day 1. This sign had resolved in one animal (No. J1) on Day 5, and in 5 animals (Nos. J8, J9, J11, J13 and J14) by Day 6 but was still present in all remaining animals at study termination.

Bodyweight: A loss in bodyweight was recorded for three females dosed at 10% v/v, three females dosed at 25% v/v (Nos. J9 to J11) and three females dosed at 50% v/v (Nos. J13, J14 and J16). All remaining animals gained weight during the study.

Applicant's summary and conclusion

Interpretation of results:

other: Substance is a skin sensitiser (1B)

Remarks:

accordance with EU CLP (1272/2008 and its amendments)

Conclusions:

The substance is a skin sensitizer (1B) in the Local Lymph node assay (OECD guideline 429).

Executive summary:

This Local Lymph Node Assay (OECD TG 429) was performed to determine the sensitising potential of the substance in mice. Groups of 4 mice were treated with test concentrations of 10, 25 and 50% v/v acetone/olive oil 4:1, or a vehicle treated control. Hexyl cinnamic aldehyde (HCA), freshly prepared as 10, 25 and 50% v/v dilution in acetone/olive oil 4:1, was used as positive control. Clinical observations and bodyweights were recorded and lymph node proliferation was determined using 3HTdR incorporation. The number of radioactive disintegrations per minute (dpm) reflect the proliferation reponse of lymph node cells, and were 3181.75, 4635.55, 11376.25, and 21482.15 dpm/animal for the vehicle control, 10%  25%, and 50% concentration groups, respectively. This corresponds with a lymph node proliferation of  n.a, 1.5, 3.6 and 6.8, respectively, for the substance-treated groups, calculated as the Stimulation Index (SI). No mortality and no signs of systemic toxicity were observed. Greasy fur was noted for all control and test animals post-dose from Day 1. This was resolved in 6 animals but was still present in all remaining animals at study termination. A loss in bodyweight was recorded for three females dosed at 10% v/v, three females dosed at 25% v/v  and three females dosed at 50% v/v. All remaining animals gained weight during the study. An EC3 value (the concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation) of 20.71% was calculated. Based on the absence of effects at the lowest concentration tested, a NOEC of 10% could be established. Under the conditions of this study, a maximum SI of 6.8 was calculated.