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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD TG Guideline 429 and GLP with a modification and without positve control substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The modification refer to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immun system
Principles of method if other than guideline:
Modified LLNA (IMDS = Integrated Model for the Differentiation of Skin Reactions): The modification refers to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune system, as also recommended in the update of OECD TG 429.

GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: red granulate
Details on test material:
Content: at least 97 %

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:
- Weight at study initiation:
- Housing: in groups of 6
- Diet ad libitum
- Water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: polyethylene glycol 400
Concentration:
0% (vehicle control); 1%, 10% and 25 % as suspension in polyethylene glycol 400
No. of animals per dose:
NMRI mice, female, 6 animals/group
Details on study design:
The test item in the formulation, the vehicle were applied epicutaneousely onto the dorsal part of both ears.of the animals. This treatment was repeated on three consecutive days. The volume administered was 25 µl/ear. The used concentrations were based on the experience with this test system and the properties of the test item.
The animals were anaestetized by inhalation of carbon dioxid and sacrificed one day after the last application. The appropriate organs were then removed .Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline
Investigations:
-weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell count of the subtancetreated lymp nodes by the vehicle treated ones
- ear swelling
- ear weight
Positive control substance(s):
not specified
Statistics:
Mann-Whitney or Wilcoxon significance test(U-test ) at levels of 5 and 1 %
Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method
In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Sheffe's method, which can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:
no data

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
It has to be clarified that the positive level for the cell count index is exclusively defined for the NMRI mice used for this study: the positive level is 1.25 for cell count index. Compared to vehicle treated animals, the measured cell counts in the test substance groups reached or exceeded the 'positive level' defined for this assay. The cell count indices were determined 1.0 (vehicle), 1.53, 1.63, 1.48 Thus, the positive level was reached or exceeded. The positive level which is 2x10[exp-2] mm increase has not been reached in all dose groups. In addition, there was no significant increase in the ear weights.
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Modified LLNA; measurement of cell counts instead of radioactive labelling.

Any other information on results incl. tables

The results show that the test item (Macrolex Rot E2G Gran.) has a weak sensitizing potential in mice after dermal application. There was an increase compared to vehicle treated animals regarding the cell counts and the weight of the draining lymph nodes in all dose groups.

Thus, a hint for a substance specific activation of the cells of the immune system via dermal application was found by the method used, i.e. the test item has a weak skin sensitizing potential.

An irritating potential at the same doses measured by acute ear swelling or ear weights, however, could not be determined.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Executive summary:

Macrolex Rot E2G was examined for sensitization according to OECD TG 429 under GLP conditions. The modification refer to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immun system, as also recommended in the update of OECD TG 429. NMRI mice were treated dermally with 0% (vehicle control); 1%, 10% and 25 % as suspension in polyethylene glycol 400 once per day on 3 consecutive days. One day later the evaluation was done: cell count index of draining lymph nodes, ear swelling and ear weight.

Compared to vehicle treated animals, the measured cell counts in the test substance groups reached or exceeded the 'positive level' defined for this assay;1.25. The cell count indices were determined 1.0 (vehicle), 1.53, 1.63, 1.48 Thus, the positive level was reached or exceeded. The positive level which is 2x10[exp-2]mm increase of ear swelling has not been reached in all dose groups. In addition, there was no significant increase in the ear weights

Thus, the results show that Macrolex Rot E2G has a weak sensitising potential in mice after dermal application.