N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Aug. 2019 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

EC Number:

204-613-6

EC Name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Cas Number:

123-26-2

Molecular formula:

C38H76N2O4

IUPAC Name:

12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Envigo RMS Spain S.L. Ctra. St. Miquel del Fai, Km. 3.5 08182 - Sant Feliú de Codines Barcelona – Spain
Health status: Specific Pathogen Free (SPF). Females will be nulliparous and non-pregnant.
Rationale for selection of species / strain: The Sprague Dawley rat is a suitable rodent strain, acceptable to regulatory authorities as a recommended rodent test system, and for which extensive background data are available.
Total number of animals: 6 animals for the first step (3 males and 3 females) and 2 spare animals. Each additional step will contain 3 males and 3 females.
Age of the animals at exposure: 8 - 12 weeks
Animals per cage: 4 (before distribution) and 3 (after distribution)
Bedding material: Sodispan (SR-CHOPO-T) (autoclavable)
Change of cages: At least once a week and when deemed necessary throughout the study period
Inclusion criteria on arrival: Veterinary inspection
Acclimatisation period: Animals will be acclimatised to Vivotecnia housing facilities at least 5 days prior exposure. Acclimatisation to the nose-only restraining tubes will be performed for at least 30 minutes the day of exposure.
Light cycle: 12h light : 12h dark
Temperature: 22 ± 3 ºC
Relative humidity: 30 – 70 %
Food: Global diet
Reference: 2914C
Supplier: Envigo
Nutritional / contaminant contents: Certificate of analysis for the batch used in the study is included in Annex III
Food availability: Ad libitum (animals were deprived of food during exposure)
Drinking water: Tap water
Watering: Bottles
Quality control: Certificate of analysis is included in Annex III
Water availability: Ad libitum (animals were deprived of water during exposure)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

2.325 µm

Geometric standard deviation (GSD):

2.27

Remark on MMAD/GSD:

Means of two measurements

Details on inhalation exposure:

Inhalation exposure was performed using a flow-past, nose-only exposure system. The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The mean flow of air at each tube was approximately 0.844 L/min, which was sufficient to minimize re-breathing of the test aerosol as it is more than twice the respiratory minute volume of rats. Exposure chambers type EC-FPC-232 (anodised aluminium, volume inside compartment: approximately 3 L), equipped with glass exposure tubes were used. The rats were individually exposed in glass tubes matching their size. Before treatment start, the homogeneity for the different levels of the exposure chamber was confirmed. The temperature and relative humidity of the test atmosphere in the exposure chamber were maintained as required by experimental conditions. Air flow was monitored regularly.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

nominal : 5 mg/l
mean dose : 4.88 mg/l

No. of animals per sex per dose:

3

Control animals:

no

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50 cut-off

Effect level:

> 4.88 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

One male animal (ID 3) was found dead during exposure. No other animals were found dead during the performance of the study.

Clinical signs:

other: Coat soiled, chromorrhinorrhea, crhromodacryorrhea, wet fur, respiratory problems (such as low respiratory rate, loud and difficult breathing and respiratory crackles) was well as apathy were recorded in all surviving animals after exposure. Some of these

Body weight:

A decrease in mean body weight was observed between study day 1 (exposure) and study day 2 in females animals and up to day 4 in males. This decrease is normally observed in nose-only exposure inhalation studies due to the stress caused by the restraining and the fasting conditions during the exposure period but could be extended due to the signs showed until day 7 of study. In general, from study day 2 in females and from day 4 in males, to the end of the observation period (day 15), body weight increased gradually in all animals.

Gross pathology:

White areas in the lungs were recorded for surviving animals. In the case of animal ID 3, that was found dead during exposure, additional black spots in the lungs and white areas in the trachea were observed. No relevant macroscopic findings were observed in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

All validity criterias are fulfilled. The study can be considered as valid. Under the study conditions, the LC50 cut-off of the test substance was considered to be >4.88 mg/l air in rats. Taking the mortality into account and according to the guideline OECD 436, the test substance is not classified as toxic by inhalativ route according to the criteria of EU CLP Regulation (1272/2008/EEC). Based on the GHS classification criteria, the test item can be considered as “Category 5”.

Executive summary:

Introduction and experimental design

The present study has been designed to evaluate the acute inhalation toxicity of the test item Bisamid (hereinafter the test item) in male and female Sprague Dawley rats by the acute toxic class (ATC) method (OECD test guideline Nº 436). This method allows the classification of the test item according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). For that purpose, a group of three male and three female Sprague Dawley rats was exposed by nose-only, flow-past inhalation to the test item at a mean concentration of 4.88 mg/L air during 4 hours. All animals were observed for clinical signs and mortality during the exposure and the subsequent 14-day observation period.Body weight was recordedjust before starting exposure, 24 h, 72 h and one week thereafter and also before sacrifice two weeks after exposure. At the end of the observation period, all surviving animals were subjected to a gross necropsy and all macroscopic abnormalities were recorded.

Results and Conclusions

The ranges of aerosol concentration, temperature, relative humidity and air flow rate were considered satisfactory for a study of this type. In addition, the test item was considered to be respirable to rats.

Coat soiled, chromorrhinorrhea, chromodacryorrhea, wet fur, respiratory problems (such as low respiratory rate, loud and difficult breathing and respiratory crackles) was well as apathy were recorded in all surviving animals after exposure. Some of these respiratory problems lasted up to day 7 of study. From study day 8 until the end of the 14 day-observation period, no clinical signs were observed in any of the surviving animals and all of them exhibited a normal behavior. A decrease in mean body weight was observed between study day 1 (exposure) and study day 2 in females animals and up to day 4 in males. This decrease is normally observed in nose-only exposure inhalation studies due to the stress caused by the restraining and the fasting conditions during the exposure period but could be extended due to the signs showed until day 7 of study. In general, from study day 2 in females and from day 4 in males, to the end of the observation period (day 15), body weight increased gradually in all animals.

White areas in the lungs were recorded for surviving animals. In the case of animal ID 3, that was found dead during exposure, additional black spots in the lungs and white areas in the trachea were observed. No relevant macroscopic findings were observed in any of the animals.

It can be concluded that, under the present experimental conditions:

-        The exposure of male and female Sprague Dawley rats to the test item for 4 hours resulted in one premature death during exposure.

-        The marginal body weight loss between study days 1 and 2 (up to day 4 for male animals) as well as the respiratory problems and apathy observed were mainly associated to the test item exposure.

-        White areas in the lungs were recorded upon gross necropsy in any of the animals.

-        Taking the mortality into account and according to the guideline OECD 436, the LC₅₀cut-off of the test item was greater than 4.88 mg/L air (gravimetric aerosol concentration).

-        Based on the GHS classification criteria, the test item can be considered as “Category 5”.