N,N'-ethylenebis[N-acetylacetamide]

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2003 to January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study and according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

GLP compliance:

yes

Test type:

fixed concentration procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenebis[N-acetylacetamide]

EC Number:

234-123-8

EC Name:

N,N'-ethylenebis[N-acetylacetamide]

Cas Number:

10543-57-4

Molecular formula:

C10H16N2O4

IUPAC Name:

N,N'-ethylenebis[N-acetylacetamide]

Details on test material:

The test substance identified as TAED,
Batch#002680,
Lot#000743-07
was received, from the Sponsor, on December 5, 2003 and was further identified with PSL Reference Number 031205-2D.
The test substance was a white powder, wasstored at room temperature and was expected to be stable for the duration of testing
(Expiration: November 4, 2004).
Documentation of the methods of synthesis, fabrication, or derivation of the test substance is retained by the Sponsor.
Prior to aero solization, the test substance was ground in a ball mill for 24 hours. The Certificate o fAnalysis for the test sample is shown as
Appendix A.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
Number of Animals: 10
Sex: 5 males and 5 females. Females assigned to the test were nulliparous and non-pregnant.
Species / Strain: Rat / Sprague-Dawley derived, albino
Age / Body Weight: Young adult (8 - 9 weeks) / males 266 - 276 grams and females 192 - 220 grams at study initiation
Source: Received from Ace Animals, Inc., Boyertown, PA on December 23, 2003

HUSBANDRY
Housing: The animals were housed singly in suspended stainless steel caging with mesh floors, which conform to the size recommendations in the
most recent Guide for the Care and Use of Laboratory Animals DHEW. Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 18 - 21°C and 30 - 52%, respectively
Photoperiod: 12 hour light / dark cycle
Acclimation Period: 7 days
Food: Purina Rodent Chow #5012 was supplied ad libitumexcept during exposure.
Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system except during exposure.
Contaminants: There were no known contaminants reasonably expected to befound in the food or water at levels that would have interfered with the results of this study. Analyses of the food and water are conducted at least once a year and the records are kept on file at Product Safety
Laboratories. The most recent analyses were conducted in October 2003 for feed and water.

IDENTIFICATION
Cage:E ach cage was identified with a cage card indicating at least the study number, dose level, and animal number and sex of the animals.
Animal: A number was allocated to each rat on receipt and a stainless steel ear tag bearing this number was attached to the rat. This number,
together with a sequential animal number assigned to study 14739, constituted unique identification.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Exposure Chamber:
Rectangular whole body Plexiglas (R) chamber with a volume of 100 liters operated under slight negative pressure.
Air Supply:
Approximately 30.1 liters per minute (Lpm) of dry filtered air from a compressed air tank (WELCO) was supplied to the dust generator. Compressed
airflow was measured with a Mass Flowmeter (Omega, Model#FMA5613). Approximately 20.6 Lpm of conditioned room air was supplied as diluent
air. Room airflow was measured with a Mass Flowmeter (Omega, Model#FMA5613). Chamber air flow was monitored throughout the exposure period and recorded periodically. Total air flow ranged from 50.4 to 50.9 with a mean of50.7 Lpm. Based on the volume of the inhalation chamber, this
airflow provided approximately 30 air changes per hour during the study.
Ambient Conditions:
The chamber temperature and relative humidity ranges during exposure were 20 - 22°C and 40 - 58% RH, respectively. The room temperature and
relative humidity ranges during exposure were 20 - 21°C and 35 - 39%RH, respectively. In-chamber and room ambient condition measurements
were made with humidity and temperature indicators (Taylor, Model 5502 and Dickson, Model TH550, respectively). Temperature and relative
humidity values were recorded every 15 minutes for the first hour of exposure and every 30 minutes thereafter.
Test Substance Preparation:
The test substance was processed in a 1.6 gallon urethane-lined milling jar (Abbethane, Paul O'Abbe) with porcelain grinding media (0.5"balls) for 24hours. After milling, the substance was sieved through a 3/8" polyethylene sieve to separate it from the grinding media and any other large particles that remained.
Dust Generation:
The test substance was packed into the dust container (Wright, Mode lDF183) and compressed to 200 lbs/in² using a lab press (Carver, Model C).
The container was then fitted with a stainless steel cutting head (Mode lDF194SS) and cutting blade (Model DF191SS) and driven by an adjustable
speed motor (Dayton, Model4Z538A). Compressed air was supplied to the dust generator at 30 psi. The aerosolized dust was then fed directly into
the chamber through the dust outlet assembly.
Gravimetric Chamber Concentration Measurements:
Gravimetric samples were withdrawn at six intervals from the breathing zone of the animals. Samples were collected using 25 mm glass fiber filters
(GF/B Whatman) in a filter holder attached by 1/4inch tygon tubing to a vacuum pump (Reliance Electric, Model#G557X). 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. The collections were carried out for 2 minutes at airflows of 4Lpm. Sample airflows were measured using a flowmeter (Omega, Model#FMA5610).
Nominal Chamber Concentration Measurement:
The amount of test substance generated and the total airflow was recorded. The nominal chamber concentration was calculated as follows and
reported.

Nominal Concentration = Total Test Substance Used / Average Airflow x Total Time

Particle Size Distribution:
An eight-stage Andersen cascade impactor was used to assess the particle size distribution of the test atmosphere. 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 aerodynamic mass median diameter and geometric standard deviation were determined graphically using two-cycle
logarithmic probit axes.
Exposure Period:
The animals were exposed to the test atmosphere for 4 hours and 9 minutes. The exposure period was extended beyond 4 hours to allow the
chamber to reach equilibrium (T99). The times for 90 and 99% equilibration of the chamber atmosphere were 4.5 and 9.1 minutes, respectively. At
the end of the exposure period, the generation was terminated and the chamber was operated for a further 16 minutes with clean air. At the end of
this period the animals were removed from the chamber. Prior to being returned to their cages, excess test substance was removed from the fur of
each animal. The day of exposure was considered Study Day 0.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

8.11 mg/l nominal concentration

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of weighing: Individual body weights of the animals were recorded on the day of test substance exposure (prior to chamber placement) and again on Study Days 7 and 14 (prior to terminal sacrifice).

- Frequency of observations: The animals were observed for mortality, signs of gross toxicity and behavioral changes prior to exposure and every
15 minutes for the first 30 minutes of exposure. Additional in-chamber animal observations were limited due to the accumulation of the test
substance on the walls of the exposure chamber. The animals were examined upon removal from the exposure chamber and at least once daily
thereafter for 14 days. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic
and central nervous systems, somatomotor activity and behavior pattern.

- Necropsy of survivors performed: yes
Gross necropsies were performed on all animals. Tissues and organs of the thoracic and abdominal cavities were examined. All rats were euthanized via CO2 inhalation on Study Day 14.

Results and discussion

Preliminary study:

Prior to initiation of the inhalation exposure, two pretest trials were conducted to establish generation procedures for achievin gas closely as
possible the targeted chamber concentration (2.0 mg/L) and desired particle size distribution (mass median aerodynamic diameter less than or
equal to four µm). In these trials, adjustments were made in pump setting in an attempt to achieve these objectives.
The exposure procedures and atomization equipment used were based on the results of pretest trial number 2, which provided a gravimetric
chamber concentration of 2.04 mg/L and a mass median aerodynamic diameter of 3.0 µm.The test substance used in trial number 2 (as well as the
full test) was ground for 24 hours in a ball mill prior to aerosolization.

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.08 mg/L air

Exp. duration:

4 h

Mortality:

All animals survived exposure to the test atmosphere.

Clinical signs:

other: In-chamber animal observations were limited due to the accumulation of test substance on th ewalls of the exposure chamber. During the first 30 minutes of exposure, clinical signs in all animals included hunched posture and hypoactivity. Ocular and/or nas

Body weight:

All animals gained body weight during the study.

Gross pathology:

No gross abnormalities were notde for the animals when necropsied at the conclusion of the 14-day post exposure period.

Applicant's summary and conclusion

Interpretation of results:

other: as the LC50 ist greater than the highest conenctration tested a definite Toxicity Category cannot be assigned.

Remarks:

Criteria used for interpretation of results: US EPA pesticides

Conclusions:

Based on the results of this study, the acute inhalation LC50 of TAED is greater than 2.08 mg/L of exposure in male and female rats.

Executive summary:

An acute inhalation toxicity test was conducted to determine the potential for TAED to produce toxicity in rats following exposure

via the inhalation route (potential route of human exposure).

After establishing the desired generation procedures during pretest trials, ten healthy rats (five males and five females) were exposed

to the test atmosphere containing approximately 2.0 mg/L of TAED for four hours. Chamber concentration and particle size distributions

of the test atmosphere were determined periodically duringt he exposure period. The animals were observed for mortality and signs

of gross toxicity and behavioral changes for a 14 -day post exposure period. Body weights were recorded prior to exposure to the

test substance and again on Study Days 7 and 14 (prior to terminal sacrifice). Gross necropsies were performed on all animals at

terminal sacrifice.

All animals survived exposure to the test atmosphere and gained body weight during the course of the study. The gravimetric chamber

concentration was 2.08 mg/L. Based on graphic analysis of the particle size distribution as measured with an Anderson Cascade

Impactor, the mass median aerodynamic diameter was estimated to be 2.9microns.

In-chamber animal observations were limited due to the accumulation of test substance on the walls of the exposure chamber. During

the first 30 minutes of exposure, clinical signs in all animals included hunched posture and hypoactivity. Ocular and/or nasal discharge

were noted in six animals upon removal from the chamber and persisted up to one hour after removal. All affected animals recovered

from the above clinical signs by Study Day 1 and, along with all other animals, appeared active and healthy over the remainder of the

14 -day post exposure period. No gross abnormalities were noted for the animals when necropsied at the conclusion of the 14 -day

post exposure period.

Based on the results of this study, the acute inhalation LC50 of TAED is greater than 2.08 mg/L of exposure in male and female rats.

Therefore, TAED falls into EPA Toxicity Category IV for inhalation toxicity.