2-Pyridinecarboxamide, 4-(4-aminophenoxy)-N-methyl-

Endpoint summary

Description of key information

Study report; OECD 429, LLNA: not sensitizing [Vohr, 2004b]

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:

Sep 2004

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)

Version / remarks:

24 April 2002

Deviations:

yes

Remarks:

- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))

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

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Strain: Hsd Win:NMRI (SPF)
- Age at study initiation: 9-10 weeks
- Mean weight at study initiation: 26-34 g
- Housing: individual, in conventional Makrolon® cages type II. Bedding used: Low-dust wood granulate from J. Rettenmaier % Söhne Füllsdorf-Fabriken, 73494 Regensberg, Germany.
- Diet: ad libitum, PROVIMI KLIBA SA 3883 maintenance diet (from Provimi Kliba
SA, CH-4303 Kaiseraugst)
- Water: ad libitum, tap water (drinking bottles)
- 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

Vehicle:

dimethylformamide

Concentration:

0, 2, 10, 50 %

No. of animals per dose:

6

Details on study design:

MAIN STUDY
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 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 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)

PRE-SCREEN TESTS: not conducted due to known properties of the test item

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Simulation Index > 3

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 weights 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.

Positive control results:

The positive control results were not reported in the present study report but are part of another study: Vohr, H.-W.: Confirmation of the function of a Local Lymph Node Assay in mice (LLNA/IMDS) with Alpha Hexyl Cinnamic Aldehyde. December 16, 2003. The results show that the test item has a clear sensitizing potential. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study.

Key result

Parameter:

SI

Value:

0.88

Variability:

9.29

Test group / Remarks:

50% dose

Remarks on result:

other: cell count index

Key result

Parameter:

SI

Value:

1.1

Variability:

14.08

Test group / Remarks:

10% dose

Remarks on result:

other: cell count index

Key result

Parameter:

SI

Value:

1.05

Variability:

29.88

Test group / Remarks:

2& dose

Remarks on result:

other: cell count index

Key result

Parameter:

SI

Value:

1

Variability:

21.47

Test group / Remarks:

Vehicle control

Remarks on result:

other: cell count index

Key result

Parameter:

SI

Value:

0.98

Variability:

14.54

Test group / Remarks:

50% dose

Remarks on result:

other: weight of draining lymph nodes

Key result

Parameter:

SI

Value:

1.25

Variability:

13.52

Test group / Remarks:

10% dose

Remarks on result:

other: weight of draining lymph nodes

Key result

Parameter:

SI

Value:

1.08

Variability:

19.78

Test group / Remarks:

2% dose

Remarks on result:

other: weight of draining lymph nodes

Key result

Parameter:

SI

Value:

1

Variability:

10.11

Test group / Remarks:

Vehicle control

Remarks on result:

other: weight of draining lymph nodes

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	1.08	1.25	0.98
Stimulation index: cell count in draining lymph nodes	1.00	1.05	1.10	0.88
Ear swelling in 0.01 mm on day 4 (index)	19.00 (1.00)	19.25 (1.01)	18.92 (1.00)	18.92 (1.00)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	11.41 (1.00)	11.96 (1.05)	11.46 (1.00)	11.39 (1.00)

The 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. The "positive level" which is 1.35 for the cell count index was never reached or exceeded in any dose group. The "positive level" for ear swelling which is 2x10-2 mm increase (i.e. about 10 % of the control values) has also not been exceeded or reached in any dose group. No substance specific effects were determined for ear weights, too.

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

The body weights of the animals were not affected by the treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

Picolinamid-Phenylether 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 no sensitizing potential in mice after dermal application. A significant difference compared to vehicle treated animals regarding the weight or cell counts of the draining lymph nodes as well as ear swelling was not reached in any case.



Thus, no hint for a substance specific or non-specific activation of the cells of the immune system via dermal application was found by the method used.

Executive summary:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with Picolinamid-phenylether in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 50% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, alpha hexyl cinnamic aldehyde, the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals.

 

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance. In this study, the test item is not a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with Picolinamid-phenylether in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 50% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, alpha hexyl cinnamic aldehyde, the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals.

 

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance. In this study, the test item is not a dermal sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results a classification according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP) is not warranted.