2,5-bis(p-toluidino)terephthalic acid

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: Guideline study (OECD TG 429)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

2,5-bis(p-toluidino)terephthalic acid

EC Number:

233-651-6

EC Name:

2,5-bis(p-toluidino)terephthalic acid

Cas Number:

10291-28-8

Molecular formula:

C22H20N2O4

IUPAC Name:

2,5-bis[(4-methylphenyl)amino]benzene-1,4-dicarboxylic acid

Details on test material:

- Name of test material (as cited in study report): Toluidinosäure
- Substance type: red
- Physical state: solid
- Stability under test conditions: stable for at least 4 h in propylene glycol at 0.1 mg/mL
- Storage condition of test material: at room temperature, protected from moisture

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 6-12 weeks (beginning of acclimatisation)
- Weight at study initiation: mean: 19.3 g
- Housing: single caged in Macrolon cages (Type I)
- Diet: pelleted standard diet (Harlan Winkelmann, Borchen, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light):12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

test item concentrations of 5, 10 and 20% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The test item could be suspended in propylene glycol at up to 20%. In other vehicles (acetone:olive oil, 4:1 (v/v), DMF, methylethyl ketone, DMSO and ethanol:water, 7:3 (v/v)) only lower concentrations of the test item could be dissolved as homogenous suspensions.

- Irritation:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed (study number 915002) in two mice (pretest excluded from Statement of Compliance). The treatment of mice with 2.5, 5, 10 and 20% test item in propylene glycol did not show any signs of servere local irritation or systemic toxicity. At the concentration of 20 % reddening of the ear could not be evaluated due to the intense red colour of the test item. However, in the main experiment the ears were not dyed by the test item

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: pooled treatment groups
- Criteria used to consider a positive response:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3 HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (S.I.).
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:
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 5, 10 and 20% (w/v) in propylene glycol. The application volume, 25 µl, was spread over the entire dorsal surface (∅∼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).
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 81 µCi/ml 3HTdR (corresponds to 20.25 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 Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
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 glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-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:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

With 10% test concentration the SI was 2.0, with 25% test concentration an SI of 4.46 was obtained. An EC3 of 16.1% was calculated.The positive control showed skin sensitiziing properties.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices of 1.3, 1.8 and 1.1 were determined with the test item (Toluidinosäure) at concentrations of 5, 10 and 20% (w/v) in propylene glycol. The EC3 values could not be calculated, since all SI's are below 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The results presented were derived from pooled groups. The values of disintegratioins per minute per lymph node were given for the control group as well as for the testing groups with the test item (Toluidinosäure) at concentrations of 5, 10 and 20% (w/v) in propylene glycol. With reference to the control group value the values determined for the test item were slightly elevated but with no dose-response trends apparent. DPM per lymph node: Control group: 513.6 5% group: 663.0 10% group: 935.8 20% group: 548.5

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Female CBA/Ca mice were subjected to test the sensitisation potential of the test item according to OECD TG 429. Due to the experimentally found Stimulation Indices < 3 the test item is not regarded as a skin sensitiser in this assay.

Executive summary:

In the study the test item dissolved 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 5, 10 and 20%.

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 1.3, 1.8 and 1.1 were determined with the test item at concentrations of 5, 10 and 20% (w/v) in propylene glycol, respectively.

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