Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 July 2005 To 01 August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
for the preliminary assay, the animals were housed in the same cages as for the main test. (considered as a minor deviation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-fluoronitrobenzene
- Substance type: Organic
- Physical state: clear brown liquid
- Analytical purity: 99.86 %
- Lot/batch No.: P150510
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 9 weeks old.
- Weight at study initiation: 20.7 +/- 0.7 g.
- Housing: Individually in disposal crystal polystyrene cages.
- Diet: adapted pelleted diet (SSNIFF Spezialdiaten GmbH, Soest, Germany), ad libitum.
- Water: tap water (filtered using a 0.22 micron filter), ad libitum.
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 30 to 70 %
- Air changes: 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12h dark/ 12h light

IN-LIFE DATES: From: 27 July 2005 To: 01 August 2005 (main test)

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10, 25, 50 and 100 %
No. of animals per dose:
4 females per dose (pooled lymph node)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item was freely soluble in acetone/olive oil (4/1, v/v). A soluion was obtained whatever the proportion in this vehicle.
- Irritation: Signs of local irritation were observed (through ear thickness measurement). Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.
- Lymph node proliferation response: not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is >= 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
- Intravenous injection of 3H-TdR and sampling of auricular lymph nodes:
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µl of 0.9 % NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
- Preparation of auricular lymph node cell suspensions and determination of proliferation:
For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 ml of 0.9 % NaCl and pellets obtained were re-suspended in 0.9 % NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 ml of 5 % (w/v) trichloroacetic acid (TCA) in purified water at +4 °C overnight. After a last centrifugation, the pellets were precipitated with 1 ml of 5% TCA. Three ml of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using bêta-scintillation counting.
The results were expressed as disintegration's/mn (dpm) per group and per node.
Stimulation Indices (SI) were calculated according to the following formula:
SI= dpm of treated group/ dpm of control group
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistic was performed.

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25 %, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI= 6.16) were noted.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.66
Test group / Remarks:
Group 2: 2-fluoronitrobenzene 5%
Remarks on result:
other: No noteworthy lymphoproliferation
Parameter:
SI
Value:
0.65
Test group / Remarks:
Group 3: 2-fluoronitrobenzene 10%
Remarks on result:
other: No noteworthy lymphoproliferation
Parameter:
SI
Value:
0.85
Test group / Remarks:
Group 4: 2-fluoronitrobenzene 25%
Remarks on result:
other: No noteworthy lymphoproliferation
Parameter:
SI
Value:
0.7
Test group / Remarks:
Group 5: 2-fluoronitrobenzene 50%
Remarks on result:
other: No noteworthy lymphoproliferation
Parameter:
SI
Value:
1.37
Test group / Remarks:
Group 6: 2-fluoronitrobenzene 100%
Remarks on result:
other: No noteworthy lymphoproliferation
Parameter:
SI
Value:
6.16
Test group / Remarks:
Group 7: HCA 25%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 80 % in each group. For more details see Table 1 in the field "Any oher information on results incl. tables".

Any other information on results incl. tables

Table 1: Study results

Treatment and concentrations Cell count
viable
Cell count
dead
viability (%) Amount of cells (x 106 cells) cellularity index Number of nodes per group dpm per group dpm per node Stimulation index (SI) increase in ear thickness (% between day 1 and day 6)
AOO
0
103 7 93.64 5.15 - 8 617.75 77.22 - - 4.90
Test item: 5 % 101 14 87.83 5.05 0.98 8 408.04 51.01 0.66 1.00
 Test item: 10% 94 12 88.68 4.7 0.91 8 404.37 50.55 0.65 -6.12
 Test item: 25% 104 21 83.20 5.2 1.01 8 525.06 65.63 0.85 -2.00
 Test item: 50% 135 12 91.84 6.75 1.31 8 432.98 54.12 0.70 2.00
 Test item: 100% 160 36 81.63 8.00 1.55 8 848.14 106.02 1.37 -8.82
HCA 25 % 235 9 96.31 23.5 4.56 8 3807.26 475.91 6.16 -

dpm: disintegration per minute

viability = viable cells/ (viable cells + dead cells) x 100

cellularity index = amount of cells (x106cells) in the treated group/ amount of cells (x106cells) in the vehicle group

stimulation index = dpm of treated group/ dpm of control group

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this test, the test item 2- fluoronitrobenzene did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the potential of the test item 2 -fluoronitrobenzene to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, 28 female CBA/J mice were allocated to 7 groups. Five treated groups of 4 animals receiving the test item 2 -fluoronitrobenzene at the concentration of 5, 10, 25, 50 or 100 %. One negative control goup of four animals receiving the vehicle (mixture acetone/olive oil (4/1, v/v)). One positive control group of four animals receiving the reference item, alpha-hexylcinnamaldehde (HCA), a moderate sensitizer, at the concentration of 25 %. During the induction phase, the test item, vehicle or refenrence item was applied over the ears (25 µl per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. The test item was freely soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained whatever the proportion in this vehicle. Consequently, the concentrations selected for the preliminary test were 10, 25, 50 and 100 %. Since the test item was non-irritant in the preliminary test , the highest concentration retained for the main test was the maximal practicable concentration (100 %). In the main test, no mortality and no clinical signs were observed during the study. No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups. No lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with the positive control (HCA) at 25 %. These results were obtained under experimental conditions acceptable regarding the purpose of the study. No deviation from the agreed study plan was observed. Under the experimental conditions of this test, the test item 2 -fluoronitrobenzene did not induce delaed contact hypersensitivity in the murine Local Lymph Node Assay.