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

Endpoint summary

Description of key information

Data summary for acute oral and inhalation endpoints.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 16 July 2020 to 11 August 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

fixed dose procedure

Species:

rat

Strain:

other: RccHan™:WIST

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Ltd
- Females nulliparous and non-pregnant: yes
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 168 to 176 grams
- Fasting period before study: overnight prior to and approximately four hours after dosing
- Housing: cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fiber bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
- Diet (e.g. ad libitum): freely available standard rodent diet (Teklad 2014C Diet)
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: at least five days
Method of randomisation in assigning animals to test and control groups: The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of one or four rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): not stated butthe animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark per 24 hours

IN-LIFE DATES: From: 16 July 2020 To: 11 August 2020.

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v aqueous methyl cellulose

Details on oral exposure:

VEHICLE (1% w/v aqueous methyl cellulose)
- Concentration in vehicle: 30 mg/L (300 mg/kg/ bw dose level) or 200 mg/L (2000 mg/kg/bw dose level)
- Amount of vehicle (if gavage): 10 ml/kg
- Justification for choice of vehicle:

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

Doses:

Sighting investigations: 300 and 2000 mg/kg body weight
Main study: 2000 mg/kg body weight

No. of animals per sex per dose:

Sighting investigations: 1 animal at 300 mg/kg body weight, 1 animal at 2000 mg/kg body weight
Main study: 4 aninals at 2000 mg/kg body weight

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of any clinical signs and the time were recorded at each observation. All animals were observed for 14 days after dosing. The weight of each rat was recorded on Days -1 (not reported), 1 (prior to dosing), 8 and 15. Individual weekly body weight changes and group mean body weights were calculated.
- Necropsy of survivors performed: yes
- Other examinations performed: body weight, macropathology

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths during the study.

Clinical signs:

other: There were no clinical signs of reaction to treatment throughout the study.

Gross pathology:

No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute median lethal oral dose (LD50) to female rats of the test substance was demonstrated to be greater than 2000 mg/kg body weight.

Executive summary:

Introduction

The acute oral toxicity of the test substance was determined using a test method designed to meet the requirements of the following guidelines:

 

• EEC Methods for the determination of toxicity and other health effects. Commission Regulation No. 440/2008, Part B, Method B.1 bis. Acute Oral Toxicity: Fixed Dose Procedure. 30 May 2008.
  
  


• OECD Guideline for Testing of Chemicals No.420 ‘Acute Oral Toxicity – Fixed Dose Method’ Adopted 17 December 2001.


• EPA Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity EPA 712‑C-02-190. December 2002.


• Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau, November 24, 2000.

 

 

Method

Fasted female rats received a single oral gavage dose of the test item, formulated in 1% w/v aqueous methyl cellulose, at the following dose levels:

 

Sighting investigations: 300 and 2000 mg/kg body weight

 

Main study: Based on the results of the sighting investigations a further four fasted females were similarly dosed at 2000 mg/kg body weight.

 

During the study, clinical condition, body weight and macropathology investigations were undertaken.

 

Results

There were no deaths during the study.

 

There were no clinical signs of reaction to treatment throughout the study. All animals were considered to have achieved satisfactory body weight gains throughout the study.

 

No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

 

Conclusion

The acute median lethal oral dose (LD50) to female rats of the test substance was demonstrated to be greater than 2000 mg/kg body weight.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

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

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

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:

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.

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.

 

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 5.06 mg/m³ air

Physical form:

inhalation: aerosol

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because skin contact in production and/or use is not likely

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The acute oral and inhalation studies did not show evidence of adverse effects, or meet the criteria for classification according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.  An acute dermal study is not required because skin contact in production and/or use is not likely.

The overall conclusion is that the substance is not classified for acute oral or inhalation effects.