7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁷,³⁸.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,27,29(45),30,32,34,36,40,43-docosaene-18,39-dione; 7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁸,³⁹.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,29,31,33,35,37(45),38,40,43-docosaene-18,27-dione

Endpoint summary

Description of key information

Skin sensitization: not sensitizing in LLNA, according OECD TG 429, GLP-compliant, 30 % test substance in acetone/olive oil, mouse, 2005, K1

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:

JuIy 26, 2005 - Sep. 19, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted April 24, 2002

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: Powder / black
- Storage condition of test material: Room temperature
- Analytical purity: 99.9%
- Expiration date of the lot/batch: unlimited at room temperature

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Age at study initiation: Ca. 6 - 8 weeks
- Weight at study initiation: 17.2 g - 19.1 g
- Housing: single housing
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 15 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30- 70%
- Air changes (per hr): fully air-conditioned rooms in which a central air-conditioning system
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: JuIy 12, 2005 (Arrival of the animals) To: Aug. 01, 2005 (Sacrifice of the animals / Determination of lymph node weight, cell count and ear weight)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

30%

No. of animals per dose:

6

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 30% preparation in AOO 4:1 was the highest applicable concentration.
- Lymph node proliferation response: The results did not show increased lymph node parameters and ear weights after application of a 30% preparation in AOO 4:1.Therefore this dose level was selected for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The parameters used to characterize the response are lymph node cell count and to a certain extent weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce Iymph node responses, the weight of ear punches taken from the area of test substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
If a test substance does not show a statistically significant and/or biologically relevant increase in cell count and/or Iymph node weight as compared to the vehicle control in the presence of statistically significantly and/or biologically relevant increased ear weights as indication of skin irritation, it is considered not to be a sensitizer.
If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance is considered to be a sensitizer (this was not the case for the test substance).
If statistically significant and/or biologically relevant increases in ear weights are running in parallel to the increase in cell count and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values may be performed. If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the
statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response (comparison with historical controls were not necessary in this study).
If a test substance does not elicit a statistical significant increase in lymph node weight and/or cell count but shows a clear concentration related increase in response, further investigating of the sensitization potential at higher concentrations should be considered (this was not the case for the test substance).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance preparation was produced on a weight by weight (w/w) basis shortly before the application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The test substance was applied as a suspension for all applications.

Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.

Application volume: 25 µL per ear

Site of application: Dorsal part of both ears

Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site

The animals were treated with vehicle or test substance preparation and were sacrificed on study day 5 by cervical dislocation.

Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal.

Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL of phosphate-buffered physiological saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.

Positive control substance(s):

other: A concurrent positive control (reliability check) with a known sensitizer was not included into this study (for details see: Any other informations on materials and methods).

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of lymph node weight, ceII count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
Lymph node weight, celI count and ear weight: WILCOXON - Test

Parameter:

other: Lymph node weight index

Value:

0.86

Variability:

1.00 (control)

Test group / Remarks:

30% in AOO 4:1

Parameter:

other: Cell count index

Value:

0.81

Variability:

1.00 (control)

Test group / Remarks:

30% in AOO 4:1

Parameter:

other: Ear weight index

Value:

1.04

Variability:

1.00 (control)

Test group / Remarks:

30% in AOO 4:1

Cellular proliferation data / Observations:

Treatment of the mice with a 30% test substance preparation induced no statistically significant increase in lymph node cell counts or lymph node weights when compared to the vehicle control group. On the 2nd and 3rd day of application and on the day of lymph node removal slight black discolored ears were noticed in all animals of the test group. Treatment of mice induced a statistically significant increase in ear weight as compared to vehicle control group which indicates some irritation of the ear at this concentration. The expected body weight gain was generally observed in the course of the study. No abnormalities were observed during general observation. Lymph node weight and cell count: Test group mean values / standard deviations / indices Lymph Node Weight [mg] 1.) vehicIe AOO 4:1: 6.0 / 1.0 / 1.0 2.) 30% in AOO 4:1: 5.2 / 0.6 / 0.86 Cell Counts [Counts/Lymph Node Pair] 1.) vehicIe AOO 4:1: 12.859.667 / 2.888.022 / 1.0 2.) 30% in AOO 4:1: 10.449.333 / 1.761.782 / 0.81 Ear weight [mg]: Test group mean values / standard deviations / indices 1.) vehicIe AOO 4:1: 28.9 / 1.0 / 1.0 2.) 30% in AOO 4:1: 30.1 / 1.0 / 1.04 # SD standard deviation # statistically significant for the value p

No signs of systemic toxicity were noticed. On the 2nd and 3rd day of application and on the day of lymph node removal slight black discoloration of the ears were noticed in all animals of the test group.

The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as a 30% preparation in AOO 4:1.

The limited, but statistically significant increase in ear weight indicates some irritation of the ear at this concentration. The test article does not have a skin sensitising effect in the Murine Local Lymph Node Assay under the test conditions chosen.

t

Mean indices (= fold of change as compared to the vehicle control):
===========================================================
* Indices * Lymph node * cell * ear *
* * weight * count * weight *
===========================================================
* vehicle AOO 4:1 * 1.00 * 1.00 * 1.00 *
* 30% in AOO 4:1 * 0.86 * 0.81 * 1.04 *
===========================================================

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of the study it is concluded, that the test article does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the non-radioactive variant of the Murine Local Lymph Node Assay based on the OECD guideline 429 and in accordance with GLP. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular Iymph nodes on repeated application of the test substance to the dorsal skin of the ears.

Groups of 6 female CBA/Ca mice each were treated with a 30% w/w preparation of the test substance (highest applicable concentration) in AOO 4:1 (mixture of acetone:olive oil Ph.Eur./DAB in a ratio 4:1 parts by volume) or with the vehicle alone. The study used 1 test group and 1 control group. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed. On the 2nd and 3rd day of application and on the day of lymph node removal slight black discoloration of the ears were noticed in all animals of the test group. The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as a 30% preparation in AOO 4:1. The limited, but statistically significant increase in ear weight indicates some irritation of the ear at this concentration. In conclusion, the test article does not have a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To investigate the skin sensitizing potential of the test substance, a local lymph node assay was conducted according to OECD 429 and under GLP conditions (BASF, 2005). Six female CBA/CaOlaHSD mice were treated by daily application of 25 µl of either 30% test substance in acetone / olive oil (4:1) or with vehicle alone to the dorsal surface of each ear for three consecutive days. The animals were sacrificed on study day 5 by cervical dislocation. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal. Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. A single cell suspension was prepared as soon as possible after dissection and the cell count was determined using a Casy®-Counter. Treatment of the mice induced a statistically significant increase in ear weight as compared to the vehicle control group which indicates some irritation of the ear at this concentration. On the 2nd and 3rd day of application and on the day of lymph node removal slight black discolored ears were noticed in all animals of the test group. Treatment of the mice with a 30% test substance preparation induced no statistically significant increase in lymph node cell counts or lymph node weights as compared to the vehicle control group.The expected body weight gain was generally observed in the course of the study. No abnormalities were observed during general observation. From the results of the study it is concluded, that the test article does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

 

Further toxicological data of category members:

This finding was supported by the data obtained for additional members of the same substance category. In three LLNA assays conducted according to OECD guideline 429 and in compliance with GLP, three additional members of the same category were analyzed for their sensitization potential. None of the tests gave a positive response, therefore all tested substances were considered as not sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No increase in the stimulation index was observed in the LLNA (OECD 429). Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.