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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 28 November to 6 December 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: in compliance with GLP and OECD guidelines (as the data is used in a read-across approach, a maximal reliability score of 2 was attributed).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: LAB 3822.
Supplier: ROQUETTE FRERES.
Batch Number: FAL 07/25.
Galenic form: slightly yellowish liquid.
Molecular weight (salt form): not applicable.
Molecular weight (base form): about 426 g/mol.
Salt/Base ratio: not applicable.
Expiry date: March 2009.
On 17.Jul.2007, 26.2 kg samples of test item were received, in vial labelled ”LAB 3822, batch No. FAL 07/25”. The Sponsor confirmed that ”LAB 3822 - Di-ester-isosorbide” in the certificate of analysis corresponds to ”LAB 3822”, name used throughout the study plan.
Storage condition: Immediately upon receipt, LAB 3822 was registered, then stored at ambient temperature in accordance with the Sponsor's instructions.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage R. Janvier - Route des Chˆenes secs - 53490 - Le Genest St Isle- France.
- Age at study initiation: Generally 8 to 12 weeks at the start of the experiment.

Female were nulliparous and non gravid.

- Weight at study initiation:
Animals treated with:
• vehicle: 21.4 g ± 0.5 g,
• test item (100%): 20.6 g ± 0.5 g,
• DNCB 0.5% (positive control): 21.1 g ± 0.6 g,
• Acetone (negative control): 21.6 g ± 0.4 g.
Animals were weighed on the day of randomisation (D-1) and the last day of the study (D6).

- Housing:Observations were performed at the time of delivery of the animals and daily during the period of acclimatisation. Animals were housed in cages of standard dimensions with sawdust bedding (SAFE, Reference B8/20). Cages were cleaned according to CERB internal SOPs.

- Diet (e.g. ad libitum): RM1 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilized) was available ad libitum except during the fasting experimental period. The criteria for acceptable levels of contaminants in the feed supplied were within the limits of the analytical specifications established by the diet manufacturer.

- Water (e.g. ad libitum):Drinking water was available ad libitum in polycarbonate feeder bottles with a stainless steel nipple. A specimen of water is obtained every 6 months and sent to the Laboratoire Départemental d'Analyse du Cher - 216, Rue Louis Mallet - 18014 Bourges Cedex, France, for analysis. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications.

- Acclimation period:Minimum of five days before treatment in the laboratory animal house where the experiment took place.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): in an air-conditioned 20-24°C
- Humidity (%): between 45% and 65%
- Air changes (per hr): 10 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
other: The test item was soluble at 75%(v/v) in acetone/olive oil. The vehicle used for the positive control item was acetone.
Concentration / amount:
Concentrations were expressed as percentage volume of test item/volume of preparation (v/v): 100% (undiluted), 75% (v/v), 50% (v/v) and 25% (v/v) were tested.
Challenge
Concentration / amount:
Concentrations were expressed as percentage volume of test item/volume of preparation (v/v): 100% (undiluted), 75% (v/v), 50% (v/v) and 25% (v/v) were tested.
No. of animals per dose:
The number of animals per group is the minimum number enabling an accurate assessment of the studied effect according to the General Requirements of OECD Guideline No. 429 (April 24, 2002).
Details on study design:
RANGE FINDING TESTS:


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: mouse received a cutaneous application over the dorsal surface of both ears (25μL) every 24 hours
- Exposure period: 3 days
- Test groups: 4 groups/16 animals
- Control group: 4 female treated with negative contol

Positive control substance(s):
yes

Study design: in vivo (LLNA)

Vehicle:
other: acetone/Olive oil (A00)
Concentration:
100% ; 75%(v/v) ; 50%(v/v) ; 25%(v/v)
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Stimulation Index (SI)



Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: Migrated information from in vivo LLNA study
Remarks on result:
other: Reading: 1st reading. Clinical observations: No clinical signs and no mortality were observed during the main study.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
expressed as the ratio of mean OD of the test nodes relative to that recorded for control (vehicle) nodes and Cellularity Index expressed as the ratio of mean amount of cells (x 10 exp 6 cells) in the treated group and the mean amount of cells (x 10 exp 6 cells) in the vehicle group.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions adopted, the test item LAB 3822 (batch FAL 07/25) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay using CBA/J female mice after three consecutive days of treatment.
Executive summary:

Possible skin sensitising potential of LAB3822 (batch FAL 07/25) was evaluated in the female mouse CBA/J using an in-vivo non-radioactive Local Lymph Node Assay (LLNA) test method in accordance with the General Requirements of OECD Guideline No. 429 (April 22, 2002) and subsequent amendments and the NIH Publication No.99-4494 dated February 1999, The Murine Local Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential of chemical compounds and the European Chemicals Bureau, Method B42.

Methods

A preliminary study was performed in order to defined the maximum concentration causing slight to moderate irritation by cutaneous application of the test item LAB 3822.

In the main study, 16 females CBA/J mice were allocated to the following six groups:

• one vehicle group of four animals receiving the vehicle (acetone / olive oil),

• one treated group of four animals receiving the test item LAB 3822 at the concentration of 100%,

• one vehicle positive group of four animals receiving the vehicle of the positive control substance (acetone),

• one positive control group of four animals receiving 2,4-dinitrochlorobenzene (DNCB) at the concentration of 0.5% (w/v) in acetone.

Each mouse received a cutaneous application over the dorsal surface of both ears (25μL) every 24 hours for 3 consecutive days (days 1, 2 and 3). Ear thickness measurements and recording of local reactions were measured before each application and just before euthanasia, in order to assess any possible irritant effect of the test item LAB 3822. Lymphoproliferative response was measured by LAB 3822 incorporation of 5-bromo-20-deoxyuridine on day 6. The results were elucidated using an Elisa System on day 8.

Results

Under the experimental conditions adopted, results obtained were as follows:

Preliminary study

The concentrations selected for the preliminary study were 25% (v/v), 50% (v/v), 75% (v/v) and 100% (undiluted).

The test item LAB 3822 was non irritant in the preliminary study. Accordingly the concentration selected for the main study was 100%.

Main study

Mortality and systemic clinical signs :

No mortality and no clinical signs were observed during the main study.

Local irritation :

At D3, a discrete erythema was noted on both ears of one animal (200705602) and on the left ear of one animal (200705599) treated with the test item LAB 3822 100%.

Lymphoproliferative effect :

The test item LAB 3822, whatever the dose tested, did not produce a stimulation index equal to or greater than 3. Consequently, the EC3 value for the test item LAB 3822 can not be calculated. Summary results are presented in the following Table 1.

Table 1 Study results - LAB 3822

 Groups  Treatment and concentrations  Viability(%)  Amount of cells  Cellularity index  Stimulation index  EC3 value  Increase inear thickness
 1 Vehicle    61 2.20  1.00  1.0  NA   +3%
 2  LAB3822 100%  70  3.20  2.14  1.4  NA  +4%
 3  DNCB 0.5 %  69  23.20  22.70  4.2  NA  14%
 4  Acetone  54  1.00  1.00  1.0  NA  -3%

NA = not applicable

Viability = (amount of viable cells / amount of total cells) x 100

Amount of cells (106 cells per node)

Increased in ear thickness (% between day 1 and day 6)

cellularity index = (amount of cells x 106 in the treated group) / (amount of cells x 106 in the vehicle group)

Stimulation index = (mean optical density in the treated group) / ( mean optical density in the vehicle group)

EC3 value = theoretical concentration resulting in a SI value of 3

Conclusion

Under the experimental conditions adopted, the test item LAB 3822 (batch FAL 07/25) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay using CBA/J female mice after three consecutive days of treatment.