Dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 JUL 2002 to 14 AUG 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 429)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

; Remark: it is mentioned that the study is based also on OECD TG406

GLP compliance:

yes (incl. QA statement)

Remarks:

according to Swiss legislation of Good Laboratory Practice

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

254-100-6

EC Name:

Dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

38720-66-0

Molecular formula:

C20H10Cl2N2O2

IUPAC Name:

1,8-dichloro-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione; 1,9-dichloro-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione; 2,9-dichloro-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione

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: 7-12 weeks (beginning of acclimatisation)
- Weight at study initiation: 17.3-21.3 g (beginning of acclimatisation)
- Housing: 4 per cage, Makrolon type -3 cages
- Diet (ad libitum): Pelleted standard Kliba 3433, batch no. 34/02 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland)
- Water (ad libitum): tap water
- Acclimation period: from 31 JUL 2002 to 6 AUG 2002, under test conditions after health examination


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1, 5, 10%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Irritation: To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 1 %, 2 . 5 %, 5 % and 1 0% (w/v) (pretest excluded from Statement of Compliance).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per Iymph node (DPM/NODE) and as the ratio of 3HTdR into lymph node cells of test Iymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation¬background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer 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:
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 %, 5 % and 10 °/0 (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (diameter 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Five days after the first topical application, all mice were administered with 250 µI of79.87 pCi/ml3H-methyl thymidine (3HTdR, equal to 20.0 µCi3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with3HTdR all mice were euthanized byintraperitoneal injection of VETANARCOL at a dose of at least 2 ml/kg body weight (equivalent to 324 mg sodium pentobarbitone/kg body weight).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymphnode cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing three 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 overnight 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 of3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background3HTdR levels were also measured in two ml-aliquots of 5 % trichloroacetic acid.The ß-scintillation counter expresses3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

TREATMENT PREPARATION AND ADMINISTRATION: The preparations were made shortly before each dosing. The test item was placed into a volumetric flask an a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

Positive control substance(s):

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

Statistics:

no data

Results and discussion

Positive control results:

Stimulation indices of 2.9, 2.6 and 7.1 were determined with the positive control substance at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). Alpha-hexylcinnamaldehyde showed an allergenic potency when tested at concentration of 25 % (w/v). Based on these results a EC3 value of 11.3% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1%: 2.4 5%: 2.0 10%: 1.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Background: 7, 8 (duplicate) control: 1593 (1585 without background) 1%: 3735 (3727 without background) 5%: 3178 (3170 without background) 10%: 2567 (2559 without background)

Any other information on results incl. tables

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of this LLNA-assay the test item was not sensitising.

Executive summary:

Three groups of four female CBA mice each were treated with the test item at concentrations of 1 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) 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 Iymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently 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 ß-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. In this study stimulation indices (S.I.) of 2.4, 2.0 and 1.6 were determined with the test item at concentrations of 1%, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v).

Based on these criteria, the test item was found to be non sensitizing. Therefore no classification is required according to Regulation (EC) No 1272/2008.