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

Endpoint summary

Description of key information

Guidelined limit study for oral and inhalation route, wich revealed no adverse effects (LD50 > 2000 mg/kg bw and LC50 cut-off >4.88 mg/l air). The physicochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin. A in vivo study for dermal route is not reasonable.

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:

21 Jan. 2019 - 22. Mar. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Slovak Academy of Sciences Dobrá Voda, Slovak Republic
- Age at study initiation: 8-9 weeks; female animals were non-pregnant and nulliparous
- Animal Health: Health condition of animals was examined by a veterinarian before initiation of the study.

ENVIRONMENTAL CONDITIONS
The animals were housed in plastic cages suspended on stainless steel racks, 3 animals per cage in a room equipped with central airconditioning. The average room temperature was maintained within the range of 22.12 ± 0.24 °C, relative humidity within 54.92 ± 2.46 %. The light regimen was set to a 12-hour light /12-hour dark cycle. Sanitation was performed according to the standard operation procedures. The laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was available ad libitum. The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodically analysed and recorded.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

VEHICLE
The required amount of the test item (according to the body weight and dose) was mixed with vehicle (Olive oil) shortly before administration. The dose of 2000 mg/kg was administered in a volume of 5 mL/kg body weight.

ADMINISTRATION:
The test item was administered in a single dose by gavage using a metal stomach tube. Animals were fasted 10-12 h prior to dosing (food but not water was withheld over-night). Following a period of fasting, animals were weighed and the test item administered. After test item administration, food was withheld for further 3-4 hours.

Doses:

2000 mg/kg bw.

No. of animals per sex per dose:

3 females per treatment step.

Control animals:

no

Details on study design:

Clinical Observation
Animals were observed individually immediately after administration of the test item and 0.5, 1, 2, and 4 hours later. Each animal was inspected daily for the next 14 days.

Body Weight
Individual weights of animals were measured immediately prior to administration of the test item and weekly thereafter. Weight differences after first and second weeks after administration were calculated and recorded.

Necropsy
All test animals were subjected to gross necropsy and the results were recorded for each animal. Examinations included: external body surface and orifices, the appearance of tissues and organs in the thoracic cavity (trachea, esophagus, heart, aorta, lungs with main stem bronchi, thymus, tracheobronchial lymph node) and in the abdominal cavity (liver, spleen, adrenal glands, kidneys, ovaries, uterus including cervix, urinary bladder, small intestine, large intestine, pancreas, stomach, mesenteric lymph nodes).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed during the study.

Clinical signs:

other: During the follow up period, no animals displayed signs of intoxication, change of health, nor any other adverse reaction.

Gross pathology:

All animals were necropsied. During necropsy, no macroscopic findings were observed.

Interpretation of results:

GHS criteria not met

Conclusions:

All validity criterias are fulfilled. The study can be considered as valid. Under the study conditions, the oral LD50 of the test substance was considered to be >2,000 mg/kg bw in rats. Therefore, the test substance is not classified as toxic by oral route according to the criteria of EU CLP Regulation (1272/2008/EEC).

Executive summary:

The purpose of the study was to evaluate the potential toxic effect of the test item “Bisamid” when administered as a single oral dose to Wistar rats. The procedure according to OECD Guideline 423 Acute Toxic Class (ATC) method was used. A limit dose of 2000 mg/kg body weight was used as a starting dose. The test item administered to 6 females at a limit dose did not cause death. No signs of toxicity were observed during the first 4 hours in females or the 14-day observation period thereafter. At the end of the study the body weights of all animals were higher than the initial body weights. A slight decrease of body weights in two animals was observed between the first and second week after administration of the test item. During necropsy, no macroscopic findings were observed. The LD50 of the test item “Bisamid” is greater than 2000 mg/kg body weight after single oral administration to Wistar rats. Therefore, the test substance is not classified as toxic by oral route according to the criteria of EU CLP Regulation (1272/2008/EEC).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

reliable without restriction

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:

22 Aug. 2019 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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)

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

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.

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”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

5 µg/m³ air

Quality of whole database:

reliable without restriction

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

CLP ((EG) Nr. 1272/2008) criteria for acute toxicity are not met. A classification is not necessary. Taking the mortality into account and according to the guideline of 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”.