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Description of key information

Short description of key information:
A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of a formulation containing 2, 10 or 50 % of the test item m-toluidine for 3 consecutive days onto both ears of the animals and yielded a negative result (Vohr 2009).

Justification for selection of skin sensitisation endpoint:
This is a guideline study and performed under GLP conditions . Therefore it is evaluated with Klimisch score 1

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modified LLNA(IMDS): Measurement of cell counts instead of radioactive labelling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions).
Modifications are authorized in the OECD TG 429 and in the Note of Guidance SWP/2145/00 of the CPMP (2001).
Information on validation of IMDS and scientific justification is given in : Vohr HW et al, Arch Toxicol 73, 501-509 (2000) and Ehling G et al, Toxicology 212, 60-68 and 69-79 (2005).
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
- Age at study initiation: 9
- Weight at study initiation: 26-31 g
- Housing: individual
- 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:
acetone/olive oil (4:1 v/v)
Remarks:
the stability of thetest item was analytically verified for up to 2 hours
Concentration:
0, 2, 10, 50 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION
The test item in the formulation and the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals . This treatment was repeated on 3 consecutive days. the volume administered was 25 µl/ear. The used concentrations were based on the experiences with this test system and the properties of the test item.
The animals were anaestetized by inhalation of carbon dioxide and sacrificed one day after the last application. The approprialte organs were then removed. Lymphatic organs (auricular lymphnodes were transferred into physiological saline (PBS).
Investigations:
-weight of lymph nodes
-cell counts in lymph nodes
-stimulation index is calculated by
dividing the absolute number of weight or cell counts of the substance treated lymph nodesby vehicle treated ones
-ear swelling
-ear weight
-body weights
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable , the values from treated groups were compared with those from the control group by a one-wayanalysis of variance (ANOVA) when the variances are considered homogeneous according to a homogenicity testing like Cochran's test. Alternatively in the variances aer considered to be heterogenous (p<=0.05) a non-parametric Kruskal-Wallis test has been used. (Kruskal-Wallis ANOVA) at significance level of 5 %. Two sided multiple test procedure 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 smalest significant differences in the meatns were calculated by Scheffel's method, which according to Sachs can be used tof both equal and unequal sizes.
Positive control results:
Alpha hexyl cinnamic aldehyd, checked in regular intervals, shows a clear sensitizing potential in the local lymph node assay (IMDS)
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
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 m-toluidin. The "positive level" which is 1.4 for the cell count index, was never reached or exceeded in any dose group (0.92, 0.98, 1.16). The "positive level" of ear swelling which is 2x10-2 mm increase, has not been reached or exceeded in any dose group.: The body weights of the animals were not affected by any treatment.
Parameter:
SI
Remarks:
Cell count index
Value:
1
Test group / Remarks:
Dose = 0% - 6 control animals
Remarks on result:
other: Modified LLNA: measurement of cell counts instead of radioactive labelling
Parameter:
SI
Remarks:
Cell count index
Value:
0.92
Test group / Remarks:
Dose = 2% - 6 animals per test group
Remarks on result:
other: Modified LLNA: measurement of cell counts instead of radioactive labelling
Parameter:
SI
Remarks:
Cell count index
Value:
0.98
Test group / Remarks:
Dose = 10% - 6 animals per test group
Remarks on result:
other: Modified LLNA: measurement of cell counts instead of radioactive labelling
Parameter:
SI
Remarks:
Cell count index
Value:
1.16
Test group / Remarks:
Dose = 50% - 6 animals per test group
Remarks on result:
other: Modified LLNA: measurement of cell counts instead of radioactive labelling

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

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Executive summary:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of a formulation containing 2, 10 or 50 % of the test item o-toluidin for 3 consecutive days onto both ears of the animals. In conclusion, the results show that the test item m-toluidin has no sensitizing potential in mice after dermal application of up to and including a 50 % concentration. In addition, no indication for a non-specific (irritant) activation was detected. Therefore, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization (Vohr 2009).

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 a formulation containing 2, 10 or 50 % of the test item m-toluidin for 3 consecutive days onto both ears of the animals.

In conclusion, the results show that the test item m-toluidin has no sensitizing potential in mice after dermal application of up to and including a 50 % concentration. In addition, no indication for a non-specific (irritant) activation was detected. therefore, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization (Vohr 2009)


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of a formulation containing 2, 10 or 50 % of the test item m-toluidine for 3 consecutive days onto both ears of the animals and yielded a negative result (Vohr 2009).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

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