Ethylene diacetoacetate

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:

other: Well performed OECD and GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene diacetoacetate

EC Number:

226-724-9

EC Name:

Ethylene diacetoacetate

Cas Number:

5459-04-1

Molecular formula:

C10H14O6

IUPAC Name:

ethane-1,2-diyl bis(3-oxobutanoate)

Details on test material:

- Name of test material (as cited in study report): Acetessigsäureglycolester

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system
Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
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: 8 - 12 weeks (beginning of treatment)
Identification: Single caging. The animals were distributed into the test groups at random and identified by cage number.
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature: 22 + 3°C
relative humidity: 30-70%
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:

4

Details on study design:

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 25, 50 and 100 % /w/v) in dimethylformamide. The application volume, 25 µl, was spread over the entire doersal surface (ca. 8 mm diameter) of each ear lobe once daily for three consecutive days. A further group of mice was trated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application, all mice were administered with 250 µl of 78.1 µCi/ml 3HTdR (corresponds to 19.5 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.

The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). 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 plastic scintillation vials with 10 ml of scintillation liquid and thoroughly mixed.

The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

At test item concentration of 5, 10, 25 % the S.I. (Stimulation index) was found to be 5.24, 7.38, 9.32 respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: At test item concentration of 25, 50 and 100 % the S.I- (Stimulation index) was found to be 1.2, 1.1, 0.99

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: contol group: 2411 DPM 25 %: 2863 DPM 50%: 2637 DPM 100%: 2382 DPM

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 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. The measured ear weights (punches) of all animals treated were recorded after sacrifice. A relevant increase in ear weights (punches) was not observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Acetessigsaureglycolester was not a skin sensitiser in this assay.

Executive summary:

In the study the test item Acetessigsäureglycolester dissolved in dimethylformamide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. An increase in the ear weights of the treated animals was also not observed. In this study Stimulation Indices (S.I.) of 1.20, 1.10, and 0.99 were determined with the test item at concentrations of 25, 50, and 100 % in dimethylformamide, respectively. The test item Acetessigsaureglycolester was not a skin sensitiser in this assay.