4,4',4''-methylidynetrianiline

Skin sensitisation

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:

2010

Report date:

2014

Materials and methods

Test guideline

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 authorised in the OECD TG 429 and in the Note for 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); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4,4',4''-methylidynetrianiline

EC Number:

208-952-0

EC Name:

4,4',4''-methylidynetrianiline

Cas Number:

548-61-8

Molecular formula:

C19H19N3

IUPAC Name:

4,4',4''-methylidynetrianiline

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, The Netherlands
- Strain: Hsd Win:NMRI
- Age at study initiation: 10 weeks
- Weight at study initiation: 29 - 40 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

The stability and homogeneity of the test item in the vehicle was analytically verified for up to 4 days.

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 or the vehicle was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
The so-called stimulation (or LLN-) index is calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 and the indices of vehicle treated animals are set to 1.00.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- 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-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) 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 Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde, checked in regular intervals, shows a clear sensitising potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Weight index of draining lymph nodes: no substance specific effects were determined. Cell count index in draining lymph nodes: 'positive level' of 1.4 was just exceeded in the high dose group (not statistically significant). Ear swelling: 'positive level' of 2x10E-2 mm increase (i.e. 10 % of control level) was not reached or exceeded in any dose group. Ear weight: no substance specific effects were determined for ear weights.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: modified LLNA; measurement of cell counts, LLN weight, ear swelling and weight instead of radioactive labelling

Any other information on results incl. tables

Results of LLNA (means for 6 animals per group)

 

Parameters investigated	Vehicle	Low (2 % in DMF)	Medium (10 % in DMF)	High (50 % in DMF)
Stimulation Index: weight of draining lymph nodes	1.00	1.21	1.22	1.12
Stimulation index: cell count in draining lymph nodes	1.00	1.19	1.37	1.42
Ear swelling in 0.01 mm on day 4 (index)	18.00 (1.00)	18.25 (1.01)	18.58  (1.03)	18.33 (1.02)
Ear weight in mg/8 mm diameter punch on day 4 (index)	12.78 (1.00)	13.42 (1.05)	12.69 (0.99)	12.76 (1.00)

* statistically significant increase (p<= 0.05)

Body weights were not affected by treatment.

A LLNA/IMDS was carried out in female NMRI mice with epicutaneous application of a formulation containing 0 %, 2 %, 10 % or 50 % of the test item 4,4',4"-methylidynetrianiline in DMF for 3 consecutive days onto both ears of the animals. The results of this study point towards a specific immunostimulating (sensitizing) potential of the test item. After treatment with 4,4',4''-methylidynetrianiline there was an increase compared to control animals regarding the cell counts of the draining lymph nodes of the high dose group (50 % test item concentration). The "positive level", which is 1.4 for cell counts, has been exceeded in this dose group. A sensitizing potential can be assumed from the increases in cell proliferation in the draining lymph nodes. On the basis of our experiences using this method the "positive level" had been set to an increase in cell count index by 0.4 (i.e. index 1.4), which has been exceeded in the high dose group. Differentiation indices (DI) calculated according to our publications (1, 2) which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction was > 1 for the high concentration (50 %) tested, i.e. 1.75. This DI value does also point to a skin sensitizing potential of the test item. The "positive level" of ear swelling which is 2x 10·2 mm increase (1, 2), i.e. more than 10 % increase in index, has not been reached or exceeded in any dose group. No substance specific effects were determined for ear weights either. The EC 1.4 value calculated is 34.0 % for this test item in DMF. In accordance with the classification proposed in the Technical Report No. 78 of ECETOC this value corresponds to a weak skin sensitizer. Based on the data collected here 4,4',4"-methylidynetrianiline has to be classified as a weak skin sensitizer. Therefore, the concentration of 10 % in DMF turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. 1. Homey, B. et al (1998): An integrated Model for the Differentiation of Chemical-Induced Allergic and Irritant Skin Reactions (IMDS). Toxicol. and Appl. Pharmacal. 153, 83-94. 2. Vohr, H.-W. et al (2000): An intralaboratory validation of IMDS: Discrimination Between (Photo )Allergic and (Photo )Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509.

Applicant's summary and conclusion

Interpretation of results:

other: weak skin sensitizer

Executive summary:

The test substance was investigated for both its skin sensitizing and irritant potential in the modified Local Lymph Node Assay (LLNA) on female mice, performed according to OECD TG 429. Concentrations of 0 % (vehicle control), 2 %, 10 % and 50 % test substance in DMF were tested. Body weights were not affected by treatment. Compared to vehicle control an increase of cell counts in the draining lymph nodes was noted for the highest test substance concentration, which was not statistically significant, however, slightly exceeded the 'positive-level' index of 1.4 (i.e. 1.42). The other parameters investigated (weight of draining lymph nodes, ear swelling and ear weight) showed no relevant changes. Under the conditions of this test 4,4',4"-methylidynetrianiline should be considered as a weak skin sensitizer. The concentration of 10 % in DMF turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.