2-methylbenzene-1,4-diyl bis{4-[4-(acryloyloxy)butoxy]benzoate}

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-10-02 to 2008-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

2-methylbenzene-1,4-diyl bis{4-[4-(acryloyloxy)butoxy]benzoate}

EC Number:

700-238-1

Cas Number:

132900-75-5

Molecular formula:

C35H36O10

IUPAC Name:

2-methylbenzene-1,4-diyl bis{4-[4-(acryloyloxy)butoxy]benzoate}

Constituent 2

Reference substance name:

4-({4-[4-(Acryloyloxy)butoxy]benzoyl}oxy)-2-methylphenyl 4-[4-(acryloyloxy)butoxy]benzoate

IUPAC Name:

4-({4-[4-(Acryloyloxy)butoxy]benzoyl}oxy)-2-methylphenyl 4-[4-(acryloyloxy)butoxy]benzoate

Details on test material:

Name: Me-3K4A2
CAS no.: 132900-75-5
Chemical name: Benzoic acid, 4-[4-[(1-oxo-2-propenyl)oxy]butoxy]-, 2-methyl-1,4-phenylene ester
Puropse: industrial chemical
Colour: beige
Physical state: powder Storage: at room temperature, protected from light
Stability in solution: water: probably stable
Special procedures: prepare freshly

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Healthy CBA/J Rj mice, female, age 8 – 12 weeks, 5 mice per test group.
The animals will be derived from a controlled full barrier maintained breeding system (SPF).
Source: Janvier, F - 53940 Le Genest-Saint-Isle
The animals will be barrier maintained (semi-barrier) in an air conditioned room
- Temperature: 22 3 °C
- Rel. humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Feeding ad libitum, Altromin 1324 maintenance diet for rats and mice - maintenance
- Free access to acidified tap water (drinking water, municipal residue control, microbiol. controlled periodically)
- The animals were kept in IVC cages, type II L, Polysulphone cages on Altromin saw fiber bedding
- Certificates of food, water and bedding are filed at testing facility
- Adequate acclimatisation period (at least 5 days)

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil (3:1 v/v)

Concentration:

12,5; 25 and 50%

No. of animals per dose:

5 per group

Details on study design:

Topical Application
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. Administration of 3H-methyl thymidine Five days after the first topical application treatment all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL.
Preparation of cell suspension
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed. The draining “auricular lymph nodes” were excised and individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C overnight for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 5 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of incorporated 3H-methyl thymidine The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: p-Phenylenediamine

Statistics:

The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitizer' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Results and discussion

Positive control results:

The positive test substance worked as expected, the DPM was 10868.8, the stimulation index was 7.0.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index at a concentration of 12.5% was 1.0. The stimulation index at a concentration of 25% was 0.7. The stimulation index at a concentration of 50% was 0.6.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Disintegrations per minute at a concentration of 12.5% were 1106. Disintegrations per minute at a concentration of 25% were 851. Disintegrations per minute at a concentration of 50% were 731

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The substance is not sensitizing to the skin under the conditions used in this LLNA test.

Executive summary:

Me3K4A2 was examined for beeing a skin sensitizer by means of the Local Lymph Node Assay (LLNA) according to OECD Guideline 429 on groups of 5 female CBA/J Rj mice. Based on the results of the preliminary test the test item Me-3K4A2 was assayed for sensitising properties at concentrations of 12.5%, 25% and 50% (v/v). The vehicle used was Acetone/Olive Oil (AOO). Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with 3H-methyl thymidine. Approximately 5 hours after the 3H-methyl thymidine injection all mice were sacrificed. The draining “auricular lymph nodes” were excised. A single cell suspension of the lymph node cells for each animal was prepared. The 3H-methyl thymidine incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration. None of the three tested concentrations of the test item reached the stimulation index of3. The stimulation index at a concentration of 12.5% was 1.0 at a concentration of 25% it was 0.7 and at a concentration of 50% it was 0.6. Weight development of all animals was within the expected range (with the exception of one animal, which lost 3 g), which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

The EC3 value (derived by linear interpolation) could not be stated, as all measure points were below the stimulation index of three. Considering the reported data of this sensitisation test it can be stated that the test item Me-3K4A2 causes no reactions identified as sensitisation, as the stimulation index was below 3.0 for each concentration tested.