N,N'-[6,13-diacetamido-2,9-diethoxy-3,10-triphenodioxazinediyl]bis(benzamide)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

8,18-dichloro-5,15-diethyl-5,15-dihydrodiindolo[3,2-b:3',2'-m]triphenodioxazine

EC Number:

228-767-9

EC Name:

8,18-dichloro-5,15-diethyl-5,15-dihydrodiindolo[3,2-b:3',2'-m]triphenodioxazine

Cas Number:

6358-30-1

Molecular formula:

C34H22Cl2N4O2

IUPAC Name:

8,18-dichloro-5,15-diethyl-5,15-dihydrocarbazolo[3',2':5,6][1,4]oxazino[2,3-b]indolo[2,3-i]phenoxazine

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Housing: single
- Diet: ad libitum
- Water: ad libitum

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

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

7.5, 15, 30 %

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irrltant test concentration or the highest technically
applicable concentration, a non-GLP pretest was performed in two mice (pre-test
excluded from Statement of Compllance ). The data showed that the highest test item
concentration, whlch could be technically used, was a 30 % suspension in acetone:olive
oil, 4:1 (v/v). Also in other vehicles tested (DMSO, DMF) a higher concentration could not
be suspended. At the 30 % suspension the treated mause did not show any signs of
systemic toxicity. Due to the intense violet colour of the test item local Irritation reactions
such as ear redness couid not be detected. No swelling of the ears was observed.
The test ltem in the main study was assayed at three consecutive concentrations 7,5, 15,
and 30 %).The top dose rs the highest technically achievable concentration whflst avoiding
systemic toxicity and excessive local irritation. No severe irritant effects were tolerated
choosing the lest concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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 incorporat1on of 3HTdR at least 3-fold or greater than that that recorded in control mice as indicated by the stimulation index
- Second, that the data are compatlble with a conventional dose response although
allowance must be made (especially at high topical concentrations) for either local
toxicity or immunologlcal 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 rlght) with different test item concentratons of 7.5, 15 and
30 % (w/v) in acetone:olive oil, (v/v). The application volume, 25 ul 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 ral~ydht vehicle alone
(control animals). A halr dryer was used to dry the ear's surface as quickly as possible to
avoid loss of test ltem applied.
Administration of 3H-Methyl Thymidine
3HMmethyl thymidine (3HTdR) was purchased from Amersham International (Amersham
product code no" TRA 310; specific actlvity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were admlnistered wlth 250 μI of
78A pCi/ml 3HTdR (corresponds to 19.6 μCi 3HTdR per mouse) by intravenous injection
via a tail vein.
Observations
In addition to the sensitising reactions the followlng observatlons and data were recorded
during the test and observation period:
Mortality / Vlabillty Daily from acclimatisation start to the termination of
in-life phase.
Body weights
Clinical signs (local I systemlc)
Prior to the first application and prior to necropsy.
Daily from accllmatisation start to the termlnation of
in-Hfe phase. Especially the treatment sites were
observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body welght tables.
A statistical analysis was conducted for assessment of the dose-response relationship,
and the EC3 value was calculated according to the equatlon
EC3 = (a-c) [(3-d)/(b-d}] + c
where EC3 is the estlmated concentration of the test item required to produce a 3-fold
increase in draining lymph node cell proliferative activity; (a, b} and (c, d) are respectively
the co-ordinates of the two pair of data lying immediately above and below the SJ. value of
3 on the local lymph node assay dose response plot

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

>= 0.5 - <= 0.7

Test group / Remarks:

7.5%, 15%, 30%

Remarks on result:

other: 7.5% 0.5 15% 0.7 30% 0.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Value:

>= 4 287 - <= 5 001.7

Test group / Remarks:

7.5%,15%, 30%

Remarks on result:

other: 7.5% 4362.4 15% 4287.0 30% 5001.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met