Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2019 - 9. May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Mean MMAD during exposure was 9.40 µm. This value was above the target range (1-4 µm). Since all the efforts were made to reduce the particle size of the aerosol during the technical trials, accord. OECD 436, the results could be considered acceptable.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

EC Number:

911-553-0

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system characterisation
Species: Rat
Strain: Hsd: Sprague Dawley® SD®
Sex: Male / Female
Colour: Albino
Supplier: Envigo RMS Spain S.L.
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 recommend ed rodent test system, and for which extensive background data are available
Total number of animals: 6 animals in a single group after distribution (3 males and 3 females)
Age of the animals at exposure: 8 - 12 weeks
Animals per cage: 3 of the same sex
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 were acclimatised to Vivotecnia housing facilities for at least 5 days prior exposure.
Acclimatisation to the nose-only restraining tubes was performed for at least 30 minutes the day of exposure.
Animal identification: Digit ink marks

Target parameters
Light cycle: 12h light : 12h dark
Temperature: 19.0 – 25.0ºC
Relative humidity: 30 – 70 %

Animal diet
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:

air

Mass median aerodynamic diameter (MMAD):

9.4 µm

Geometric standard deviation (GSD):

> 1.5 - < 3

Remark on MMAD/GSD:

Mean Mass Median Aerodynamic Diameter (MMAD) of particle size distribution during exposure was calculated from two gravimetric measurements PSD #1 and PSD #2. Mean MMAD during exposure was 9.40 µm. This value was above the target range (1-4 µm).
The aerosol achieved showed a constant and small Geometrical Standard Deviation (GSD) which proves that the aerosol was consistent and the MMAD depended exclusively on the original particle size of the product that was 43 µm according to the information supplied by the sponsor. Since all the efforts were made to reduce the particle size of the aerosol during the technical trials, and according to the OECD guideline 436, the results could be considered acceptable.

Details on inhalation exposure:

Characterization of the test item atmosphere during exposure
In order to characterize the test atmosphere and to check the reproducibility of aerosol generation and dilution, several analytical parameters were determined at defined intervals during exposure.

Determination of the nominal aerosol concentration
The test item usage was determined once per exposure by weighing the amount of the test item before and after exposure to determine the quantity used. The weight used was then divided by the total air-flow volume to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.

Gravimetric determination of the aerosol concentration
Gravimetric determination of the aerosol concentration was performed twice each hour of exposure. Test aerosol samples were collected onto a Whatman filter (grade F319.04) using a filter sampling device. The sampling flow was similar to the air flow rate per exposure port. The duration of sampling was 10 minutes. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.

Determination of particle size distribution
The particle size distribution was determined gravimetrically twice during exposure. The cumulative particle size distribution of the test aerosol was determined using a PIXE cascade impactor. The particle size distribution of the test item in the generated aerosol was measured by gravimetry analyzing the test item deposited on each stage of the cascade impactor.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 4 μm for the MMAD and 1.5 to 3 for the GSD.

Determination of the temperature
The temperature in the chamber was measured continuously during exposure using a thermohygrometer (TFA 30.5036.13). The target range was 19-25 ºC. The results were reported approximately hourly from the start of the inhalation exposure.

Determination of relative humidity
The relative humidity in the chamber was measured continuously during exposure using a thermohygrometer (TFA 30.5036.13). The ideal range recommended by the OECD guideline Nº 436 is 30-70 %, although it is also indicated that these values may be unattainable under certain conditions and this was the case of this study in which compressed air (dry air) was used to dilute the test item aerosol. The results were reported approximately hourly from the start of the inhalation exposure.

Determination of oxygen and CO2 concentration
The oxygen and CO2 concentration of the test atmosphere were measured during the exposure period using a portable gas detector (PGAS-24-O2/CO2, PGAS-24). These concentrations were maintained above 19 % and below 1 % respectively. The results were reported approximately hourly from the start of the inhalation exposure.

Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. The actual airflow rate was monitored at least hourly during exposure. The target range was 1.0 ± 0.5 L/min through each inhalation tube.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Technical trials were performed without animals to investigate the conditions for aerosol generation, which included the determination of target concentration and/or technical limit. Several tests were performed to establish the highest stable aerosol concentration achievable that could be maintained at least for 4 hours using:
Different powder aerosol generators (Palas rotating brush generator and TOPAS SAG 410 aerosol generator) under varying conditions.
Different pressures for the generator air flow
Different flow rates for the dilution air flow (using the Palas generator).
The outcome of the key experiments was as follows:
Initially, a moving belt TOPAS dust generator was tested but the generator was blocked (even with desiccated test item) after just a few minutes The powder was aerosolized then by a rotating brush generator (Palas, Palas GmbH). An aerosol at a concentration of up to approximately 8 mg /L was achieved. However, the aerosol could only be generated for less than 1 hour due to the observed test item accumulation in the transfer li ne.
In another attempt, the dilution air flow was increased with the aim to reduce the test item accumulation in the transfer line by increasing the linea r velocity of the aerosol and consequently to obtain a better aerosol stability. No improvement was observed.
At a lower aerosol concentration of approximately 1 mg/L, however, a stable aerosol could be achieved although the particle size was above criteria due to the inherent properties of the test item..
In conclusion, the technical limit for the aerosol concentration was considered to be approximately 1 mg/L air. The concentration was achieved with the PALAS Solid Particle Dispenser RGB 2000

No. of animals per sex per dose:

A total of 3 male and 3 female rats (technical limit test dose, group A) were exposed for 4 consecutive hours

Control animals:

no

Details on study design:

Mortality and premature sacrifice
The animals were examined daily for mortality and morbidity. Any clinical signs, discomfort and mortality were recorded in accordance with the humane endpoints guidance document of the OECD.

Clinical observations
Clinical observations in response to treatment were performed on all animals hourly during exposure (only grossly abnormal signs), immediately and 1 h after exposure, and once daily thereafter until the end of the observation period. All animals were observed for a period of 14 days after administration. Any visible clinical signs and discomfort were recorded.

Body weight
All animals were weighed on the day of treatment just before starting exposure (study day 1), on study days 2, 4, 8 and immediately before sacrifice on study day 15.

Statistics:

No statistical analysis was required.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.05 mg/L air

Based on:

test mat.

Mortality:

All animals survived the 14-day observation period. No relevant findings were observed upon gross necropsy.

Clinical signs:

other: All animals, regardless of sex, presented wet fur up to 1 hour after exposure. Chromorrhinorrhea and chromodacryorrhea was also observed after exposure in all animals except for male ID 1. Additionally, immediately after exposure, loud breathing was recor

Body weight:

A decrease in mean body weight was observed for both males and females between study day 1 (exposure) and study day 2.
From study day 2 to the end of the observation period (day 15), body weight increased gradually in all animals.

Gross pathology:

No relevant macroscopic findings were observed in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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 no premature death for any of the animals.

The marginal body weight loss between study days 1 and 2 in all animals was considered to be mainly due to the stress related to the nose-only exposure procedure, although a contribution of the treatment with the test item cannot be excluded.

Based on the technical limitations for concentration under which the study was performed, i.e. maximum achieved concentration of 1.05 mg/L air, the LC50 of the test item could not be estimated. Since no animals died during study, the LC0 was greater than 1.05 mg/L air (gravimetric aerosol conc entration).

Due to the technical limitation in the aerosol concentration, a classification based on the GHS criteria was not applicable. Since no animal died during the study and the clinical observations recorded were not relevant and most likely caused by nose-only restraining tube stress, the test item could i n any case be estimated as “unclassified” under human health hazard.

Executive summary:

Introduction and experimental design

The present study has been designed to evaluate the acute inhalation toxicity of the test item N-(hydroxymethyl) stearamide (hereinafter the test item) in male and female Sprague Dawley rats by the protocol described in the OECD Guideline Nº 436: Acute Inhalation Toxicity. This method allows the classification of the test item according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.

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 1.05 mg/L air during 4 hours as a technical limit test.

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

Several tests were performed during technical trials to achieve the concentration of 5 mg/L. Among others, the use of two different powder aerosol generators, different pressure and flow conditions, desiccation of the test item. A stable aerosol could not be obtained at 5 mg/L in any of the tests, thus, 1 mg/L air was considered as the technical limit for a reproducible and stable aerosol.

The ranges of aerosol concentration, temperature, relative humidity and air flow rate were considered satisfactory for a study of this type. In addition, although the particle size of the aerosol was above limit criterion, it was considered to be acceptable based on the nature of the test item.

All animals survived the 14-day observation period. No relevant findings were observed upon gross necropsy.

All animals, regardless of sex, presented wet fur up to 1 hour after exposure. Chromorrhinorrhea and chromodacryorrhea was also observed after exposure in all animals except for male ID 1. Additionally, immediately after exposure, loud breathing was recorded in the male animal ID 2.

From study day 2 until the end of the 14 day-observation period, no clinical signs were observed in any of the animals and all of them exhibited a normal behavior.

A decrease in mean body weight was observed for both males and females between study day 1 (exposure) and study day 2.

From study day 2 to the end of the observation period (day 15), body weight increased gradually in all animals.

No relevant macroscopic findings were observed in any of the animals.