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

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 30 April 2021 to 9 June 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 433 (Acute Inhalation Toxicity: Fixed Concentration Procedure)

Deviations:

yes

Remarks:

A different method of identification was used for the animals than stated in the protocol (microchip identification was used in the study). This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

fixed concentration procedure

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

Test animals

Species:

rat

Strain:

other: RccHan® :WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Source: Male and female WISTAR RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
-Age at study initiation: approximately 8 – 10 weeks.
- Weight at study initiation: 255g to 263g for males and the female was 184g
- Fasting period before study: No
- Housing: The animals were housed one animal per cage during the sighting study and five of one sex per cage during the main study. The cages were made of a polycarbonate body with a stainless steel mesh lid. Autoclaved softwood bark-free fibre was used as bedding and changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum (except during exposure)
- Water (e.g. ad libitum): ad libitum (except during exposure)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not specified, but the animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

4.4 µm

Geometric standard deviation (GSD):

2.65

Remark on MMAD/GSD:

MMAD/GSD values given are for the main study.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through nose-only chamber
- Method of holding animals in test chamber: Plastic nose-only restraint tube
- Source and rate of air (airflow): In-house compressed air system (breathing quality) with a rate of airflow of 14L/minute.
-- System of generating particulates/aerosols:
Device used: Mechanical grinder
Burst time: 15 seconds
Number of bursts: Four
Sieve mesh size: 600 µm
- Method of particle size determination: Cascade Impactor, Marple 290 Series (298 configuration)
- Treatment of exhaust air:
- Temperature, humidity, pressure in air chamber: The observed chamber temperatures for all groups remained within the recommended range (19 – 25°C) for inhalation exposure of rats. All of the observed relative humidity values were below the recommended range (30 - 70%) this was considered to be a result of the dried air supplied to the chamber.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Based on preliminary study using one male and one female rat.

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

5.06 mg/L (main study)

No. of animals per sex per dose:

sighting study: 1 male and 1 female.

Main test: 5 males.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days (or other?)
- Frequency of observations and weighing:
- Necropsy of survivors performed: yes/no
- Clinical signs including body weight
- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other:

Results and discussion

Effect levels

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

> 5.06 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths during the study.

Clinical signs:

other: Clinical signs of wet fur were considered associated with the inhalation exposure procedure and a consequence of tube restraint and not related to treatment with the test substance.

Body weight:

On the day following exposure (Day 2) body weight loss was evident in all males exposed to 5.06 mg/L. The group mean body weight was comparable to Day 1 values by the next weighing occasion (Day 4) and continued to increase for the remainder of the observation period.


The changes in body weight gain on Day 2 are considered to be a consequence of the exposure (duration and removal of food and water) and therefore not test-item related.

Gross pathology:

The macroscopic examination after a single exposure followed by a fourteen day observation period for males exposed to 5.06 mg/L revealed no abnormalities.

Any other information on results incl. tables

Chamber Atmosphere Conditions

Summary data are presented below:

Group	Target aerosol concentration (mg/L)	Mean achieved aerosol concentration (mg/L)	Particle Size
			MMAD (µm)	GSD
1*	5.0	4.96	4.4	2.46
2	5.0	5.06	4.4	2.65

* The achieved aerosol concentration for Group 1 was a time-weighted average
MMAD = Mass median aerodynamic diameter
GSD = Geometric standard deviation

 

The time-weighted average achieved concentration was 99% of target for Group 1, and the mean achieved average achieved concentration was 101% of target for Group 2.

 

The test guideline states that MMAD should be ≤ 4 µm whenever possible. In this case the achieved MMAD values were slightly above the ideal size. Actions taken during preliminary generation trials to try to reduce the MMAD included altering the airflow to the generator and micronizing the test item. This did not offer any improvement to the MMAD, it is therefore considered the obtained MMAD was the smallest that could be practically obtained at the target aerosol concentration.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Category 5/unclassified, according to the Globally Harmonised System (GHS; UNITED NATIONS).

Conclusions:

N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) was administered to rats by a nose only, single exposure for 4 hours, followed by a 14-day observation period. There were no test-item related deaths, clinical signs, macropathology findings or effects on body weight.

Executive summary:

Introduction

The study was performed to assess the acute inhalation toxicity of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) to the rat.  The test method was designed to be compliant with the following test guideline:

• OECD Guideline for Testing of Chemicals No.433 “Acute Inhalation Toxicity: Fixed Concentration Procedure.” Adopted 25 June 2018.

 

Method

One male and one female received a single 4-hour nose only exposure of the test item at the target concentration of 5 mg/L and were observed for 7 days. Based on the results of this sighting investigation, a further five males were similarly exposed for the main study at the target concentration of 5 mg/L and observed for 14 days. During the study, clinical condition, body weight and macropathology investigations were undertaken.

 

Results (main study)

The mean average concentration data are summarised as follows:

Group	Mean achieved concentration (mg/L)	Particle Size
		MMAD (µm)	GSD
2	5.0	5.05	4.4

 

The mean achieved average achieved concentration was 101% of target for Group 2. The achieved MMAD values were slightly above the ideal size. Actions taken during preliminary generation trials to try to reduce the MMAD included altering the airflow to the generator and micronizing the test item. This did not offer any improvement to the MMAD, it is therefore considered the obtained MMAD was the smallest that could be practically obtained at the target aerosol concentration.

 

There were no unscheduled deaths or clinical signs during the detailed weekly physical examination. The clinical sign of wet fur is associated with the inhalation exposure procedure and was a consequence of tube restraint.

 

There were no test item-related effects on body weight and no macropathology findings were seen.

 

Conclusion

Under the conditions of this study, the (LC50) 4-hour of the test substance was in excess of 5.06 mg/L for male rats. The test substance is considered to be Category 5/unclassified, according to the Globally Harmonised System (GHS; UNITED NATIONS).