Methyl acetoacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2008 until January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance with the corresponding OECD-/EU-testing guidelines

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other 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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acetoacetate

EC Number:

203-299-8

EC Name:

Methyl acetoacetate

Cas Number:

105-45-3

Molecular formula:

C5H8O3

IUPAC Name:

methyl 3-oxobutanoate

Test material form:

other: non-viscous clear liquid

Details on test material:

- Physical state: Colourless liquid
- Storage condition of test material: Room temperature, protected from light
- Stability in solvent: > 72 h in DMSO and ethanol at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan LAboratories BV, 5960 AD Horst, The NEtherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 21.8-23.0 grams priotr treatment with 3HTdR
- Housing: single housing
- Diet (e.g. ad libitum): Pelleted standard diet, Harlan Lab. GmbH, 33178 Borchen, Germany
- Water (e.g. ad libitum): Tap water
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C (+/- 3°C)
- Humidity: 30-70%
- Photoperiod: artificial light 06:00 a.m. - 06:00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Doses of 25, 50 and 100& (in DMSO) were applied.

No. of animals per dose:

4 animals per dose group

Details on study design:

Administration and exposure:
For determination of the highest non-irritant and technically applicable test item concentration, a pretest was performed on two mice with concentrations of 50 and 100 % (w/v). The top dose of the test item is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. Four female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radiolabelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methylthymidine measured in a ß-scintillation counter.

Data and Observations:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). In addition to the sensitising reactions the following observations and data were recorded: mortality/viability once daily, body weights prior to the first application and prior to necropsy, clinical signs (local/systemic) daily from start to the termination of in-life phase.

Positive control substance(s):

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

Statistics:

The mean values amd statndard deviations were calculated in the body and ear weight tables.

Results and discussion

Positive control results:

Results of the GLP Positive Control:
- Experiment performed in July 2008.
- Positive control substance: Hexylcinnamaldehyde
- Vehicle: acetone: olive oil (4+1)
- Results: at dose concentrations of 5, 10 and 25% (w/v), stimulation indices of 5.24, 7.38 and 9.32 were observed
The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

Test concentration 25%

Remarks on result:

other: A calculation of the EC3 value was not possible since none of the tested concentrations induced a S.I. greater than 3.

Results 2

Key result

Parameter:

SI

Value:

1.91

Test group / Remarks:

Test concentration 50%

Remarks on result:

other: A calculation of the EC3 value was not possible since none of the tested concentrations induced a S.I. greater than 3.

Results 3

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

Test concentration 100%

Remarks on result:

other: A calculation of the EC3 value was not possible since none of the tested concentrations induced a S.I. greater than 3.

Any other information on results incl. tables

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG ^a)	number of lymph nodes	DPM per lymph node ^b)	S.I.
---	BG I	33	---	---	---	---
---	BG II	37	---	---	---	---
---	1	4571	4536	8	567.0
25	2	6966	6931	8	866.4	1.53
50	3	8719	8684	8	1085.5	1.91
100	4	3198	3163	8	395.4	0.70

 

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number

of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Methyl acetoacetate was not a skin sensitiser under the described conditions.

Executive summary:

The LLNA-study was performed in 2008/2009 under GLP. In order to study a possible contact allergenic potential of Methyl acetoacetate, three groups each of four female mice were treated daily with the test item at concentrations of 25, 50, and 100 % (w/v) in dimethylsulfoxide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylsulfoxide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed. An increase in the ear weights of the treated animals was also not observed. In this study Stimulation Indices of 1.53, 1.91, and 0.70 were determined with the test item at concentrations of 25, 50, and 100 % in dimethylsulfoxide. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.