N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase:16 December 2020 to 19 January 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

EC Number:

951-985-7

Molecular formula:

C30H38N6O3

IUPAC Name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

Test material form:

solid: crystalline

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/CaCrl strain mice were supplied by Charles River (UK) Ltd, Margate, UK.).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 17 to 20g. Individual body weights were within ±20% of the mean body weight for mice on the study.
- Housing: Animals were housed in groups of up to five during acclimatisation and in pairs. From day 1 the preliminary study animal was individually housed from and the main study animals were housed in pairs. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK).
. - Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 - 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least fifteen per hour
Photoperiod (hrs dark / hrs light): 12 hours in light, 12 hours in darkness

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

The vehicle was chosen because it produced the highest suitable concentration (25% w/v) out of the vehicles listed in the protocol.

Concentration:

Control = vehicle only, low dose = 5 % w/v test substance, mid-dose = 10% w/v test substance, high dose = 25% w/v test substance

No. of animals per dose:

Four per dose group.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The positive control article produced a Stimulation Index of 4.48, demonstrating adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.29

Test group / Remarks:

5 % w/v

Results 2

Key result

Parameter:

SI

Value:

ca. 1.72

Test group / Remarks:

10 % w/v

Results 3

Key result

Parameter:

SI

Value:

ca. 2.112

Test group / Remarks:

25% w/v

Any other information on results incl. tables

Mortality

There were no deaths on the study.

Clinical Signs

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 5, 10 or 25% w/v formulations of the test article.

 

The vehicle and test formulation application sites remained free of irritation.

 

Greasy fur to the back of the ears and neck were observed in all Group 5 animals from Day 1 to Day 5 indicative of the positive control vehicle being acetone/olive oil (4:1 (v/v)).

Body Weights

There was no indication of a treatment related effect on body weight.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result from the Local Lymph Node Assay, the test substance was concluded not have the potential to cause skin sensitisation.

Executive summary:

Introduction

This study was conducted to assess the potential of the test article, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide), to cause skin sensitisation in the mouse. The method used was deigned to meet the requirements of the following guidelines:

• OECD Guidelines for Testing of Chemicals Method 429 (adopted 22 July 2010).


• Method B42 of Council Regulation (EC) No 440/2008

 

Method

Following a preliminary screening test using a 25% w/v formulation, the test article was prepared for administration at 5, 10 or 25% w/v in dimethyl sulphoxide (DMSO).

Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle control, positive control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated ³H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices and the threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

Results

	Concentration of Test Article in Applied Formulation (% w/v)
	5%	10%	25%
Stimulation Index	1.29	1.72	2.12

 

The positive control article produced a Stimulation Index of 4.48, demonstrating adequate performance of the assay.

Conclusion

The test substance, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) did not induce skin sensitisation in the Local Lymph Node Assay and is considered not to be a skin sensitser.