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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP; guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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): N-Methylaniline (NMA)
- Physical state: liquid
- Analytical purity: 98.5%
- Lot/batch No.: 2010395

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.0 g - 21.5 g
- Housing: Makrolon cage type II, one animal per cage, Dust-free wooden bedding, enrichment: PLEXX mouse tunnel, nest building material Nestlets NES 3600
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air condition, 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully airconditioned rooms
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
10%, 25% and 50% of test substance in vehicle
No. of animals per dose:
5
Details on study design:
Groups of 5 female CBA/J mice each were treated with 10%, 25% and 50% w/w preparations of the test substance in MEK or with the vehicle alone. The high concentration was selected based on the presence of systemic toxicity and mortality in a pre-test using the undiluted test
substance.
The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces 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 into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was
determined in order to obtain an indication of possible skin irritation.
A concurrent positive control (reliability check) with a known sensitizer was not included in this study. Studies using the positive control substance
Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect
sensitizing compounds under the test conditions chosen.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0%: 1.00 10%: 1.72 25%: 1.30 50%: 1.38
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0%: 143.4 +/- 49.2 10%: 246.6 +/- 80.3 25%: 186.7 +/- 73.6 50%: 198.3 +/- 55.9

Any other information on results incl. tables

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Test

Group

Treatment

 

³H-thymidine

incorporation

Stimulation Index

Cell Count

Stimulation Index

Lymph Node

Weight

Stimulation Index

 

Ear Weight

Stimulation Index

1

vehicle MEK

1.00

1.00

1.00

1.00

2

10% in MEK

1.72 #

1.01

1.12

0.99

3

25% in MEK

1.30

1.01

1.12 #

0.96

4*

50% in MEK

1.38

1.24 #

1.22 #

0.96

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

* Calculation on basis of 4 animals due to an irregularity during 3H-thymidine injection.

The animals treated with the 50% test-substance concentration showed slightly reduced activity until about 30 minutes after each application. No other signs of systemic toxicity were noticed during the observation period. When applied as 50%, 25% and 10% solution in MEK the test substance did not induce biologically relevant increases in the 3H-thymidine incorporation (no increase above the cut off Stimulation Index of 3) into the cells from the auricular lymph nodes or in lymph node cell counts (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). There was no relevant increase in lymph node weights, as well. However, statistically significant increases were noted for 3H-thymidine incorporation in test group 2, lymph node cell counts in test group 4 and lymph node weights in test groups 3 and 4. All test-substance preparations did not cause any increase in ear weights, demonstrating the absence of ear skin irritation. Thus it is concluded that N-Methylaniline (NMA) does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
N-Methylaniline (NMA) did not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.