2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). The IMDS was validated and published with scientific justification in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

IDMS LLNA: Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

Principles of method if other than guideline:

This study is performed according to OECD TG 429. As stated in OECD TG 429 besides the classical radioactive method ‘other endpoints for assessment of the number of proliferating cells may be employed’ as so-called ‘me-too’ tests, if the required performance standards are fulfilled, they are ‘based on similar scientific principles and measure or predict the same biological or toxic effect’ and they are validated.
Here, an alternative method is used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes (IMDS LLNA; Integrated Model for the Differentiation of Skin Reactions). In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitizing properties. Information on validation of the IMDS LLNA 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).
In the IMDS LLNA stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify skin sensitization) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).

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-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)phenoxy]-N-methylpyridine-2-carboxamide

EC Number:

608-209-4

Cas Number:

284461-73-0

Molecular formula:

C21 H16 Cl F3 N4 O3

IUPAC Name:

4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)phenoxy]-N-methylpyridine-2-carboxamide

Constituent 2

Reference substance name:

2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-

IUPAC Name:

2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-

Constituent 3

Reference substance name:

4-{ 4-[( {[ 4-chloro-3-(trifluoromethyl)phenyl]amino }carbonyl)amino ]phenoxy }-Nmethyl-2-pyridinecarboxamide

IUPAC Name:

4-{ 4-[( {[ 4-chloro-3-(trifluoromethyl)phenyl]amino }carbonyl)amino ]phenoxy }-Nmethyl-2-pyridinecarboxamide

Details on test material:

- Purity not specified

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win:NMRI (SPF)
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 26-33 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

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 were 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 25µl/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 then 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)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)

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 the Mann-Whitney or the Wilcoxon signigicance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5 % (one-tailed for LLNA/IMDS or PNLA (larger)). Outlying values in the LN/ear weights or LN cell counts were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differentes 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 showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

cell count index

Value:

0.87

Variability:

±18.14

Test group / Remarks:

2% test item

Results 2

Key result

Parameter:

SI

Remarks:

cell count index

Value:

0.93

Variability:

±13.36

Test group / Remarks:

10% test item

Results 3

Key result

Parameter:

SI

Remarks:

cell count index

Value:

0.79

Variability:

±24.32

Test group / Remarks:

50% test item

Cellular proliferation data / Observations:

No significant dose-dependent increase in the stimulation indices for the weight or cell counts as well as for ear swelling or ear weights.

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated	Vehicle control	Dose 2 %	Dose 10 %	Dose 50 %
Stimulation index: weight of draining lymph nodes	1.00	0.84	0.88	0.83
Stimulation index: cell count in draining lymph nodes	1.00	0.87	0.93	0.79
Ear swelling in 0.01 mm on day 4 (index)	18.17 (1.00)	17.92 (0.99)	18.00 (0.99)	17.92 (0.99)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	12.54 (1.00)	12.73 (1.02)	12.80 (1.02)	13.18 (1.05)

The mice did not show any significant dose-dependent increase in the stimulation indices for the weight or cell counts as well as for ear swelling or ear weights. Slight significant decrease compared to vehicle treated animals regarding cell counts was detected in the highest dose group. This decrease is in the normal range of variance for these parameters and is such of no biological relevance. The reason for this decrease is not known but could be due to osmosis in the tissues and/or cytotoxicity induced by a relative high local concentration of the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

BAY 43-9006 was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 50 % formulated in dimethylformamide were tested. The results show that the test item has neither an irritating nor a sensitizing potential in mice after dermal application.

Executive summary:

The test item BAY 43-9006 was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 50 % formulated in dimethylformamide were tested.

The cell count indices for test item concentrations of 2, 10, and 50% were 0.87, 0.93, and 0.79, respectively. The “positive level” indicating sensitizing potential, which is 1.35 for the cell count index, was never reached or exceeded in any dose group. The “positive level” of ear swelling indicating irritating potential, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was also not reached or exceeded in any dose group.

The results show that the test item has neither an irritating nor a sensitizing potential in mice after dermal application. The validity of the assay was demonstrated by the positive results of the positive control group (alpha hexyl cinnamic aldehyde).