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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 may 2010 - 30 june 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
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-nitroaniline
- Analytical purity: 99.9 %

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain CBA/J
- Source: Charles River laboratories, 97633 Sulzfeld
- Age at study initiation: 7-12 weeks
- Weight at study initiation: ca. 15 - 25 g
- Housing: single housing in Makrolon cages, type II
- Diet: Kliba diet, Provimi Kliba SA, Basel Switzerland
- Water: ad libitum tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 % (limit dose based on pre-test)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A 50 % test substance preparation in AOO was the highest attainable test substance preparation.
- Irritation: not irritating
- Lymph node proliferation response: No relevant increase in ear weights and lymph node weights in the pre-test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The cell proliferation induced in the draining lymph nodes (auricular lymph nodes) is used for evaluating the skin sensitizing potential. 3H-thymidine incorporation and the cell count which was determmined after cell isolation from both ear lymph nodes indicated this proliferation. In addition, ear weights and lymph node weights were determined. Especially the ear weights as surrogate for ear swelling were used as an indicator for ear swelling.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL per ear, applied epicutaneously to the dorsal part of both ears, on three consecutive days.
On study day 5, the mice were injected i.v. with 20 µCi 3H-thymidine in 250 µL sterile saline into the tail vein.
Positive control substance(s):
not specified

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Test group SI= 1.03
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control Group: 1,516.2 [DPM/Lymph Node Pair] Test group: 1,562.6 [DPM/Lymph Node Pair]

Any other information on results incl. tables

EXPERIMENTAL RESULTS

Cell Counts
Test Group Treatment [Counts/Lymph Node Pair] Stimulation Index
control group vehicle AOO 8,161,333 1
test group 50% in AOO 9,560,000 1.17
³H-thymidine incorporation
Test Group Treatment [DPM/Lymph Node Pair] Stimulation Index
control group vehicle AOO 1,516.2 1
test group 50% in AOO 1,562.6 1.03
Lymph Node Weight
Test Group Treatment [mg/Lymph Node Pair] Stimulation Index
control group vehicle AOO 5 1
test group 50% in AOO 5.2 1.04
Ear Weight
Test Group Treatment [mg/animal] Stimulation Index
control group vehicle AOO 29.3 1
test group 50% in AOO 30.9 1.05

Body weights:

The expected body weight gain was generally observed in the course of the study.

Other findings:

Orange discolored urine was observed in all animals of test group 2 from day 1 up to day 5. No other abnormalities were observed during general observation.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
2-Nitroaniline does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of 2-Nitroaniline was assessed using the radioactive Murine Local Lymph Node Assay following OECD guideline 429 and in compliance with GLP. Groups of 5 female CBA/J mice each were treated with a 50% w/w preparation of the test substance in AOO or with the vehicle alone. The 50% preparation was the highest homogeneously producible test-substance concentration. The study was carried out as a limit test, using 1 test group and 1 control group. Each test animal was applied with 25 μL per ear of the test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 μCi of

3H-thymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and 3H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

Besides orange discolored urine, which was observed from day 1 up to day 5 in all animals of test group 2, no signs of systemic toxicity were noticed. When applied as 50% preparation in AOO, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well. Concomitantly, the increase of 3H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3). The test-substance preparation caused no relevant increase in ear weights. Thus it is concluded that 2-Nitroaniline does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.