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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
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP but without concurrent positive control
Qualifier:
according to guideline
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 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.

GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 28 - 35 g
- Age at the start of treatment: 10 weeks
- Housing: singly during study
- Diet ad libitum
- Water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
other: A/OO = aceton / olive oil (4:1)
Concentration:
0% (vehicle control), 2 %, 10 %, 50 %,
No. of animals per dose:
6
Details on study design:
The test item in the formulation, or 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 subtance-treated lymph nodes by the vehicle treated ones
- ear swelling
- ear weight
-body weight
Positive control substance(s):
other: no concurrent positive control substance;
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by one-way Analysis of Vartiance (ANOVA) when the variances are considered homogenous according to a homogenicity testig like Cochran's test. Alternatively, if the variances are considered to be heterogenous a non-parametric Kruskal-Wallis test has been used at significance levels of 5 %.. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. 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, wich can be used for both equal and unequal sample sizes.
Positive control results:
no data
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
It has to be clarified that the positive levels are exclusively difined for the NMRI mice used for this study: Compared to vehicle treated animals, none of the parameter measured in the test substance groups i.e. cell counts, weights of the draining lymph nodes, ear weights and ear swelling reached or exceeded the 'positive level' defined for this assay. The cell count indices were determined 1.0 (vehicle), 0.81, 1.12, 1.17. Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using alpha hexyl cinnamic aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor EL/ physiological saline solution 2 % v/v) at concentrations of 3 %, 10 % and 30 %. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study (Vohr, AT00361, April 14. 2003). A similar check is done in regular intervals using one of the above mentioned vehicles in order to confirm the reliability of the method. The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5 %, 10 % and 40 % clearly showed the sensitizing potential of the test item (Vohr, PH38176, August 26, 2014).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; mearurement of cell counts instead of radioactive labelling.

The positive level of ear swelling which is 2x10E-2 mm increase , i.e. about 10 % of the control values, has not been reached or exceeded in any dose group.of the test item.

Body weights of the animals was not affected by any treatment.

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

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of formulation

containing 0% (vehicxle control), 2 %, 10 %, or 50 % of the test item o-toluidin diluted in aceton / olive oil (4:1) for 3 consecutive days onto both ears of the animals. The study was conducted according to OECD TG No. 429 and No. 406 under GLP condititions

Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using alpha hexyl cinnamic aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor EL/ physiological saline solution 2 % v/v) at concentrations of 3 %, 10 % and 30 %. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study (Vohr, AT00361, April 14. 2003). A similar check is done in regular intervals using one of the above mentioned vehicles in order to confirm the reliability of the method. The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5 %, 10 % and 40 % clearly showed the sensitizing potential of the test item (Vohr, PH38176, August 26, 2014). . The result show no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 50 % concentration of o-toluidin. Additionally, no indication for a non-specific(irritant) activation by the test item o-toluidin was detected.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of formulations.

containing 0% (vehicxle control), 2 %, 10 %, or 50 % of the test item o-toluidin diluted in aceton / olive oil (4:1) for 3 consecutive days onto both ears of the animals. The study was conducted according to OECD TG No. 429 and No. 406 under GLP condititions

Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using alpha hexyl cinnamic aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor EL/ physiological saline solution 2 % v/v) at concentrations of 3 %, 10 % and 30 %. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study (Vohr, AT00361, April 14. 2003). A similar check is done in regular intervals using one of the above mentioned vehicles in order to confirm the reliability of the method. The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5 %, 10 % and 40 % clearly showed the sensitizing potential of the test item (Vohr, PH38176, August 26, 2014). The result show no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 50 % concentration of o-toluidin. Additionally, no indication for a non-specific(irritant) activation by the test item o-toluidin was detected.

Migrated from Short description of key information:
o-Toluidin has no sensitizing potential in mices after dermal application in the Local Lymph Node Assay (LLNA/IMDS) according to the respective guideline

Justification for selection of skin sensitisation endpoint:
There is a study available which is performed according to the respective guideline under GLP conditions. No concurrent positive control is included, but the reliability of the test is proved by an additional study with a positive control substance which is cited in the report. Therefore the study is evaluated with Klimisch score 2

Justification for classification or non-classification

On the basis of this finding o-toluidine has not to be classified according to Directive 67/548/EEC or according to Regulation (EC) No. 1272/2008