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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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: guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
The modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain Crl:NMRI BR (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item 1,3-Dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione.
The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Vehicle:
dimethylformamide
Concentration:
Group 1 Vehicle (DMF)
Group 2 2% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF)
Group 3 10% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF)
Group 4 50% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF)
Group 5 30% Alpha Hexyl Cinnamic Aldehyde (in DMF)
No. of animals per dose:
6 animals/test item group and 6 control animals
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A modified Local Lymph Node Assay (IMDS) was carried out in mice. The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. Tabular summary of the LLNAlIMDS results 1. Direct LLNA (NMRI mice, female, 6 animals/group) Groups Weight index Cell count index (index of mean +/-SD in 0/0) Gr. 1 1.00 +/-22.25 1.00 +/-23.22 Gr. 2 1.01 +/-18.33 1.03 +/-62.60 Gr. 3 0.93 +/-22.57 0.97 +/-27.26 Gr. 4 1.19 +/-16.16 1.24 +/-24.50 Gr. 5 1.57s +/-19.95 1.78s +/-30.28 2. Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm) Groups day 1 day 4 Index day 4 (mean +/-SD in %) Gr. 1 17.42 +/-2.96 17.67 +/-3.69 1.00 Gr. 2 16.75 +/-3.71 17.42 +/-4.55 0.99 Gr. 3 17.17 +/-4.18 17.50 +/-4.56 0.99 Gr. 4 17.42 +/-3.84 18.75 +/-6.48 1.06 Gr. 5 17.92 +/-3.73 22.67s +/-16.65 1.28 3. Ear weight (NMRI mice, female, 6 animals/group, in mg per 8 mm diameter punch) Groups day 4 Index day 4 (mean +/-SO in %) Gr. 1 11.84 +/-8.37 1.00 Gr. 2 11.11 +/-6.69 0.94 Gr. 3 10.93 +/-6.22 0.92 Gr. 4 12.01 +/-7.48 1.01 Gr. 5 14.66s +/-14.90 1.24 s = statistically significant increase (p< 0.05) Group 1 Vehicle (DMF) Group 2 2% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF) Group 3 10% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF) Group 4 50% 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (in DMF) Group 5 30% Alpha Hexyl Cinnamic Aldehyde (in DMF)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Based on results obtained in validation studies and general experiences with this test system groups of mice were treated with vehicle, 2 %, 10 % or 50 % 1 ,3-Dihydro-4( or 5)-methyl-2H-benzimidazole-2-thione in DMF.

The NMRI mice did not show an increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item 1 ,3-Dihydro-4( or 5)-methyl-2H-benzimidazole-2-thione.

The "positive level", which is 1.4 for the cell count index, was never reached or exceeded in any dose group.

The "positive level" of ear swelling, which is 2x 10E-2 mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group.

In the high dose group deposits of the test item were standing at the ears of one animal (no. 24), in places.

After treatment with Alpha Hexyl Cinnamic Aldehyde (group 5) the NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, has clearly been exceeded.

The "positive level" of ear swelling, which is 2x10E-2 mm increase, i.e. about 10 % of the control values, has been exceeded in the positive control group. This increase was of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was detected, too.

It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbreed mice used for this study Such positive limits have to be calculated for each strain of mice individually.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

The modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/ test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of

the test item 1 ,3-Dihydro-4( or 5)-methyl-2H-benzimidazole-2-thione.

A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde was included.

The study was conducted with the following test item concentrations:

Test item: 0 % (vehicle control), 2 %, 10 % and 50 %. Positive control: 30 % Alpha Hexyl Cinnamic Aldehyde

The test item was formulated in dimethylformamide (DMF) to yield a solution in the low and mid dose and a suspension in the high dose. The positive control was formulated in DMF to yield a solution.

Compared to vehicle-treated animals, none of the parameters measured in the substancetreated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay [6, 8, 9]. These results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % 1,3-Dihydro-4(or 5)-methyl-2H-benzimidazole-2- thione in this test system.

In conclusion, these results show that the test item 1,3-Dihydro-4(or 5)-methyl-2Hbenzimidazole- 2-thione was not sensitising in mice after dermal application of up to and including a 50 % concentration. No indication for a non-specific (irritant) activation was

detected, too. Therefore, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

These results are verified by the comparison with the results of the positive control group (Alpha Hexyl Cinnamic Aldehyde).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Compared to vehicle-treated animals, none of the parameters measured in the substance treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. These results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % 1,3-Dihydro-4(or 5)-methyl-2H-benzimidazole-2- thione in this test system.


Migrated from Short description of key information:
A modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/test item group and 6 control animals) to determine if there is a sensitizing potential of the test item 1 ,3-Dihydro-4( or 5)-methyl-2H-benzimidazole-2-thione.
The modification of the assay by measuring the cell counts instead of radioactive labeling provides comparable sensitivity. A further modification was done by including the measurement of the ear swelling after treatment leading to a much more simplified and reliable assay (Integrated Model for the Differentiation of Skin reactions (IMDS). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN; cell counts / LN weights) with the immediate unspecific acute skin reaction (ear swelling / ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested.

Justification for selection of skin sensitisation endpoint:
key study used

Respiratory sensitisation

Endpoint conclusion
Additional information:

no data


Migrated from Short description of key information:
no data

Justification for classification or non-classification

Due to the negative result of the LLNA a classification for skin sensitisation is not justifed.