Bis(16-methylheptadecyl) malate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 15 Jun 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

yes

Remarks:

no analytical purity/batch not reported

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): diisostearyl malate
- Physical state: viscous colourless liquid
- Analytical purity: no data
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 9 weeks
- Weight at study initiation: 21.5 ± 1.3 g
- Housing: The animals were housed individually in disposable crystal polystyrene cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary test: 2.5, 5, 10 and 25%
Main test: 1, 2.5, 5, 10, 25%

No. of animals per dose:

1 (preliminary test)
4 (main test)

Details on study design:

RANGE FINDING TESTS
- Compound solubility: highest concentration that was suitable for dosing: 25%
- Irritation: neither signs of systemic toxicity nor local skin irritation were observed up to the highest concentration of 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3Hmethyl-thymidine incorporation
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a “non-sensitiser”.

TREATMENT PREPARATION AND ADMINISTRATION
25 μL of the test item was applied to the dorsal surface of each ear of each mouse for three consecutive days; Further groups of four mice each received the vehicle or positive control alone in the same manner. Local irritation reactions were assessed. On day 6 an injection of 250 μL 0.9% NaCl containing 20 μCi of 3-Hmethyl-thymidine (3HTdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised and pooled for each experimental group. For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at 4 °C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3HTdR using β-scintillation counting. The results were expressed as disintegration's/min (dpm) per group and per node. Stimulation Indices (SI) were calculated according to the formula: SI = dpm of treated group / dpm of control group.

Positive control substance(s):

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

Statistics:

Not applicable

Results and discussion

Positive control results:

A group of four animals was treated with 25 μL of the positive control substance α-hexylcinnamaldehyde (HCA) as a 25% solution in acetone/olive oil 4/1 (v/v). The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is 6.72. Therefore, α-hexylcinnamaldehyde (HCA) was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Test material concentrations of 1, 2.5, 5, 10 and 25% in acetone/olive oil (4/1 v/v) show stimulation indices (SI) of 1.21, 1.14, 1.32, 0.87 and 1.39, respectively. Based on this result, the test substance is considered as a non-sensitiser under the experimental conditions of the test.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The number of disintegrations per minute/node (dpm/node) for the vehicle was 45.30. Test material concentrations of 1, 2.5, 5, 10 and 25% in acetone/olive oil (4/1 v/v) show dpm/node of 54.93, 51.55, 59.87, 39.63 and 62.87, respectively.

Any other information on results incl. tables

Table 1: Experimental results of the LLNA assay

Concentration (% in v/v) acetone/olive oil (4/1)	dpm/group	dpm/node	Stimulation index	Result
Vehicle	362.40	45.30	NA	NA
1	439.41	54.93	1.21	Negative
2.5	412.42	51.55	1.14	Negative
5	478.97	59.87	1.32	Negative
10	317.01	39.63	0.87	Negative
25	502.98	62.87	1.39	Negative
Positive control	2436.56	304.57	6.72	Positive

dpm: disintegration per minute

Systemic clinical signs and mortality

No clinical signs and no mortality were observed during the study.

 

Body weight

The body weight change of the treated animals was similar to that of the control animals.

 

Local irritation

No cutaneous reactions and no increase in ear thickness were observed at any tested concentration.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified