2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Remarks:

The acute inhalation toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2015 to 30 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1989

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF 12-Nousan-8147

Version / remarks:

1999

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

452-330-3

EC Name:

-

Cas Number:

314020-40-1

Molecular formula:

C14H20N2O2

IUPAC Name:

2-(2,6-diethyl-4-methyl-phenyl)propanediamide

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 ANIMALS
- Source: SAGE® Labs
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 333-386 g (males), 218-250 g (females)
- Fasting period before study: No
- Housing: Singly housed in suspended stainless steel caging which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): Envigo Teklad Global 16% Protein Rodent Diet® #2016 ad libitum
- Water (e.g. ad libitum): Municipal water ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 43-58
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 h cycle

IN-LIFE DATES: From: 08 December 2015 To: 30 December 2015

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.15 µm

Geometric standard deviation (GSD):

2.22

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only inhalation chamber, the base unit terminates the chamber with a 0.5-inch diameter tube for discharged air.
- Exposure chamber volume: 6.7 L
- Method of holding animals in test chamber: Animals were individually housed in polycarbonate holding tubes which seal to the chamber with an "O" ring during exposure.
- Source and rate of air: Air compressor, 36.0 Lpm
- Method of conditioning air: Filtered generator air was supplied to the spray atomization nozzle by an air compressor, and measured with a Mass Flow Controller. Additional filtered mixing air from the same air compressor, measured with a Mass Flow Controller, was introduced into the chamber to help uniformly distribute the test atmosphere by creating a vortex at the chamber inlet. Chamber airflow was monitored throughout the exposure period and recorded periodically. The exposure was conducted under slight negative pressure.
- System of generating particulates/aerosols: The test substance was aerosolized using a modified Wright Dust Generator. The test substance was packed into the dust container and compressed 1000 lbs/in^2 using a lab press. The container was then fitted with a cutting head. Compressed generator and mixing air were supplied to the dust generator. The aerosolized dust was then fed directly into the chamber through the dust outlet assembly.
- Method of particle size determination: An eight-stage 1 ACFM Andersen Ambient Particle Sizing Sampler. Samples were withdrawn from the breathing zone of the animals at two intervals. The filter paper collection stages were weighed before and after sampling to determine the mass collected upon each stage. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were determined graphically using two-cycle logarithmic probit axes.
- Temperature, humidity, pressure in air chamber: 20-21°C, 35-39%
- Compressed generator / mixing air: 30/30 psi
- T90/T99: 0.43 min / 0.86 min


TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric samples were withdrawn at 6 intervals from the breathing zone of the animals. Samples were collected using 37 mm glass fiber filters (Whatman™ GF/B) in a filter holder attached by ¼ inch Tygon® tubing to a vacuum pump. Filter papers were weighed before and after collection to determine the mass collected. This value was divided by the total volume of air sampled to determine the chamber concentration. Sample airflows were measured using a Mass Flow Controller.
- Samples taken from breathing zone: yes

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

5.16 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:Observed for mortality during the exposure period. Examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube and at least once daily thereafter for 14 days. Individual body weights of the animals were recorded prior to test substance exposure (initial) and again on Days 1, 3, 7, and 14 (terminal).
- Necropsy of survivors performed: yes

Statistics:

Not applicable (limit test, no mortalities).

Results and discussion

Preliminary study:

Prior to initiation of the full inhalation study, pre-test trials were conducted to establish generation procedures to achieve, to the extent possible, the desired chamber concentration (5.0 mg/L) and desired particle size distribution (mass median aerodynamic diameter between 1 and 4 μm). The procedures and aerosolization equipment used in the full test were based on the results of pre-test trial number 3 which provided a gravimetric chamber concentration of 5.20 mg/L and a mass median aerodynamic diameter of 2.25 μm.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.16 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals survived.

Clinical signs:

other: Following exposure all rats exhibited irregular respiration. In addition, one male was hypoactive. All animals recovered by Day 1 and appeared active and healthy for the remainder of the 14-day observation period.

Body weight:

All animals gained body weight during the study.

Gross pathology:

No gross abnormalities were noted.

Any other information on results incl. tables

Table 1: Summary of acute study test atmosphere characteristics of CA3250

Test atmosphere characteristics
Parameter	Target concentration (mg/L)
	5.0
Gravimetric concentration	5.16±0.11 mg/L (n=6)
Nominal concentration	9.49 mg/L
Particle size MMAD; GSD	2.12, 2.17 µm; 2.19, 2.25
	(at 1.5 and 3 hours into exposure respectively)
Particle size distribution	% total particles captured (by weight)
Size range (µm)	Run 1 (1.5 hours into exposure)	Run 2 (3 hours into exposure)
Particles<9.0 µm (% w/w)	2.4	3.2
Particles < 5.8 µm (% w/w)	5.4	5.6
Particles< 4.7µm (% w/w)	6.5	7.2
Particles<3.3 µm (% w/w)	19.0	18.9
Particles < 2.1 µm (% w/w)	26.6	27.5
Particles< 1.1µm (% w/w)	25.1	21.1
Particles<0.7 µm (% w/w)	8.4	7.8
Particles < 0.4 µm (% w/w)	3.5	5.0
Particles < 0 µm (% w/w)	3.2	3.6
Average total air flow	36.0 L/min
Air changes / hour	322
Temperature (exposure chamber)	20-21°C
Humidity (exposure chamber)	35-39%
T99	0.86 m

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the single exposure acute inhalation LC50 of the test substance is greater than 5.16 mg/L in male and female rats.

Executive summary:

An acute inhalation toxicity test was conducted with rats to determine the potential of the test substance to produce toxicity from a single exposure via the inhalation (nose-only exposure) route.

After establishing the desired generation procedures during the pre-test trials, ten healthy rats (5/sex) were exposed to the test atmosphere for 4 hours. Chamber concentration and particle size distributions of the test atmosphere were determined periodically during the exposure period. The animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for up to 14 days following exposure. Body weights were recorded prior to exposure (initial) and again on Days 1, 3, 7, and 14 (terminal). Necropsies were performed on all animals at terminal sacrifice.

The gravimetric chamber concentration was 5.16 mg/L. The average mass median aerodynamic diameter was estimated to be 2.15 µm based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Particle Sizing Sampler with an average geometric standard deviation of 2.22.

All animals survived exposure to the test atmosphere and gained body weight during the study. Following exposure, all rats exhibited irregular respiration. In addition, one male was hypoactive. However, all animals recovered by Day 1 and appeared active and healthy for the remainder of the 14-day observation period. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

Therefore, under the conditions of this study, the single exposure acute inhalation LC50 of the test substance is greater than 5.16 mg/L in male and female rats.