Registration Dossier

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-06-17 till 2008-07-16
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Details on test material:
- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.5-22.4g
- Housing:single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Species: CBA/CaOlaHsd

- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

test item at concentrations of 5, 10, and 25% in dimethylformamide
No. of animals per dose:
Details on study design:
The highest test item concentration, which can be technically used was a 25 % suspension in dimethylformamide.
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 25% on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. Due to the colour of the test item, redness of the ear skin could not be

- Name of test method:
OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 24 April 2002).

- 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.

The test item in the main study was assayed at 5, 10, and 25%. The top dose is the
highest technically achievable concentration whilst avoiding systemic toxicity and
excessive local

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Mean and standard deviation

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Concentration S.I. 5: 1.78 10: 1.84 25 4.87
other: disintegrations per minute (DPM)
Remarks on result:
other: concentration DPM control: 5328 5: 9506 10: 9807 25: 25980

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the colour of the test item, redness of the ear skin could not be observed.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation and results of individual data

 Test Item concentration % (w/v)  Group  Measurement DPM   Calculation         Result
       DPM-BG a)    number of lymph nodes DPM per lymph node b)   S.I
---  BG I  191  --- ---  ---  --- 
--- BG II  33  ---  ---  --- --- 
---  3688  3556  8 444.5   
 5 36636  36524  4565.5   10.27 
 10 40203  40091  5011.4   11.27 
25  15893  15781  1972.6   4.44 

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are above 3

Applicant's summary and conclusion

Interpretation of results:
Migrated information
The test item test material was found to be a skin sensitiser under the described conditions.
Executive summary:

In the study the test item test material suspended in dimethylformamide 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 25%.

All treated animals survived the scheduled study period and no signs of toxicity were observed. Due to the colour of the test item, redness of the ear skin could not be observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 10.27, 11.27, and 4.44 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide. The EC3 value could not be calculated, since all obtained SI´s were above 3.