Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-09-29 to 2005-10-18

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:

2005

Report date:

2005

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:

Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

EC Number:

228-528-9

EC Name:

Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Cas Number:

6289-46-9

Molecular formula:

C10H12O6

IUPAC Name:

1,4-dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): 3,6-Dihydro-2,5-dihydroxyterephthalicacid dimethylester

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Study design: in vivo (non-LLNA)

No. of animals per dose:

4

Positive control substance(s):

not required

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0.0, 1.25 , 2.5, 5.0% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a pretest was performed in two mice with concentrations of 0.625, 1.25, 2.5 and 5% (w/v). The data showed that the highest test item concentration, which could be technically used was a 5 % suspension. At this concentration the treated mouse did not show any signs of irritation.


MAIN STUDY
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled;
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.



TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
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 1.25, 2 5 and 5% (w/v) in propylene glycol. The application volume, 25 µl, was spread over the entire dorsal surface (0-8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-Methyl Thymidine
3H-methyl thymidine: specific activity of 2 Ci/mmol; concentration, 1 mCi/ml
Five days after the first topical application, all mice were administered with 250 µl of 78.5 µCi/ml 3HTdR by intravenous injection via a tail vein.
Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental.
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 ceils 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 glass scintillation vials with 10 ml of 'Ultima Gold' 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 p-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)

Statistics:

not applicable

Results and discussion

Positive control results:

positive

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1.25 % : 1.03 2.50 % : 1.10 5.00 % : 1.02

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0.00 % : 2949 1.25 % : 3049 2.50 % : 3255 5.00 % : 2999

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

not a skin sensitiser

Executive summary:

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 1.25, 2.5 and 5%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.. In this study Stimulation Indices (S.I) of 1.03, 1.10 and 1.02 were determined with the test item at concentrations of 1.25, 2.5 and 5% (w/v) in propylene glycol, respectively. The test item 3,6-Dihydro~2,5-dihydroxyterephthalicacid dimethylester was not a skin sensitiser in this assay.