Dipotassium dihydrogen ethylenediaminetetraacetate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 6, 2015 to January 21, 2016

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dihydrogen ethylenediaminetetraacetate

EC Number:

217-895-0

EC Name:

Dipotassium dihydrogen ethylenediaminetetraacetate

Cas Number:

2001-94-7

Molecular formula:

C10H14N2O8.2K

IUPAC Name:

dipotassium dihydrogen 2-({2-[bis(carboxylatomethyl)amino]ethyl}(carboxylatomethyl)amino)acetate

Specific details on test material used for the study:

Test Material Name: PRIVENTZ™ Liquid
Trademark: PRIVENTZ™ Liquid
Lot/Reference/Batch Number: 3D0301D601
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the active ingredient concentration at 38.46% wt. by turbidimetric titration and having a pH of 5.27 and specific gravity of 1.231 (Pearson, 2015).
Test Material Stability Under Storage Conditions: The test material safety data sheet lists a storage stability of twenty-four months.

Test animals

Species:

rat

Strain:

other: F344/DuCrl

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: F344/DuCrl rats. Selection of this strain and species was based on a variety of considerations including hardiness, low incidence of respiratory disease, general acceptability for inhalation toxicity studies, and availability of historical control data.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: Animals were seven weeks at arrival and ten weeks at the time of exposure.

Physical and Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology
and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed two or three per cage in stainless
steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate environmental conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study. Animals were acclimated to the nose cones for at least two hours on the day preceding exposure to the test material.

Housing:
After assignment, animals were housed two or three per cage in stainless steel cages. Cages had solid floors with corncob bedding and a nylon bone for enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
The following environmental conditions were maintained in the animal rooms.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Note: Photoperiod times may change due to study-related activities.

Randomization and Identification:
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the
start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water:
Feed and municipal water was provided ad libitum except during the nose-cone acclimation period the day prior to exposure and during the 4-hour exposure period. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained at Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use
Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study were Acute Tox 01, Humane Endpoints 01, and Animal ID
01.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.17 µm

Geometric standard deviation (GSD):

2.19

Details on inhalation exposure:

Route, Method of Administration, Frequency, Duration and Justification:
Inhalation was selected as the route of administration since it is a potential route of human exposure. The test material was administered via a single 4-hour nose-only exposure.

Stability:
The test atmosphere was generated and immediately delivered to the test system; therefore analysis of the stability of the test material under the conditions of administration was not necessary for this study.

Solubility
A 1:1 dilution of PRIVENTZTM Liquid in water was prepared immediately prior to exposure by stirring on a stir plate until in solution. The test material is completely miscible in water, the solvent used in PRIVENTZTM Liquid. The liquid aerosol test atmosphere was generated and immediately delivered to the nose-only inhalation exposure system.

Chambers:
A 42-liter, Dow-modified ADG nose-only chamber [30 centimeters (cm) in diameter by 60 cm high] was used for the study. Compressed filtered air supplied to the chamber was at ambient temperature. Airflow through the chamber was determined with a manometer which measured the pressure drop across a calibrated orifice plate and was maintained at approximately 49 liters per minute, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 70 air changes per hour. The manometer was calibrated with a gas meter (Model DTM-115, Singer Aluminum Diaphragm Meter, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. The chamber was operated at a slightly negative pressure relative to the surrounding area and was contained within a secondary vented area. Chamber and exposure room temperature were recorded from two thermocouples attached to an electronic digital thermometer (Control Company, Friendswood, Texas), one thermocouple extended into the exposure chamber and the second was stationed next to the chamber. Chamber relative humidity was monitored by a hygrometer (VWR, West Chester, Pennsylvania) stationed in the interior of the chamber. Based on the 49 liter per minute flow rate, the theoretical equilibrium time to 99% (T99) of the target concentration was 3.9 minutes. The animals were placed on the chamber after the T99 had elapsed and were removed after 240 minutes of continuous exposure.

Generation System:
A liquid aerosol of PRIVENTZ™ Liquid was generated by metering the test material with a FMI pump (Fluid Metering, Inc., Oyster Bay, New York) into a stainless steel ¼-J spray nozzle (Spraying Systems Co., Wheaton, Illinois). The test material was mixed
with compressed air in the spray nozzle and aerosol was sprayed into the chamber. Since the formulation contained materials of varying vapor pressures, the test material was not recycled.

Exposure Conditions:
Exposure room temperature, chamber temperature, humidity and airflow were monitored continuously and recorded approximately every 30 minutes during the exposure period.

Exposure Concentration:
The concentration of the test material was determined gravimetrically 4 times during the exposure period. Samples were taken by drawing air, at 1 L/minute, through a sample probe located in the breathing zone of the animals. Aerosol particles were
collected on preweighed 0.45 micron polytetrafluoroethylene (PTFE) laminated 47 mm filters (PALL Corporation, Ann Arbor, Michigan). A substantial portion of the exposure chamber atmosphere consisted of vapor (primarily the test material solvent vehicle, water), therefore, vapors present were collected on two preweighed silica sorbent tubes (SKC Inc., Eighty Four, Pennsylvania) placed downstream of the filter. The total mass of aerosol and vapor collected during each sampling interval was determined by subtracting the pre-sample mass of the filter and sorbent tubes from the post-sampling weights. Prior to test material exposure, a chamber background measurement, with animals on the chamber, was taken. The time-weighted average (TWA) exposure concentration of the test material was calculated from the combined mass of aerosol (filter) and vapor (sorbent tubes) collected during each sample period, minus the chamber background, times the dilution factor (0.5).

The efficiency and performance characteristics of the gravimetric sampling train were assessed before preliminary chamber aerosol samples were measured. Initially, a known mass of un-diluted test material was deposited (spiked) onto the filter and a known volume of air was pulled through the sample train (filter and two silica sorbent tubes) to simulate sampling from the exposure chamber. The performance of the sample train was determined to be greater than 98% efficient by calculating the average of 3 samples.

The nominal concentration was calculated based on the amount of test material fed into the generation system divided by the total chamber airflow.

Particle Size Determination:
The aerodynamic particle size distribution was determined twice during the exposure period by drawing samples from within the animal breathing zone, at a flow rate of 3 liters/minute for 3 minutes using a constant flow air sampling pump through a multi-stage
mercer-style cascade impactor. The MMAD and geometric standard deviation (GSD) were determined for each sample as well as the average of the samples.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Time-weighted average chamber concentration of 5.80 mg PRIVENTZ™ Liquid per liter of air.

No. of animals per sex per dose:

Five rats/sex/dose

Control animals:

no

Details on study design:

Animal Observations and Criteria of Response:
A cage-side examination was conducted at least once a day, generally at the same time each day (usually in the morning). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that
were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that would be observed include, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Cage-side observations, in which only positive findings were documented, are summarized with clinical observations.
Animals were weighed and examined prior to exposure to the test material and observed at least every 30 minutes during the exposure period. All rats were weighed on test days 2, 4, 8, 11, and 15 during the two-week post-exposure period.
All rats were submitted for a complete necropsy examination on test day 15. Non-fasted rats submitted alive for necropsy were anesthetized with a mixture of isoflurane vapors and medical oxygen, and were then placed in a CO2 chamber to continue anesthesia. While under anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a trained technologist qualified to recognize common lesions. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The head was split longitudinally to facilitate examination of the nasal passage. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from
the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. Tissues were not saved and histopathologic examination was not
performed unless deemed meaningful on selected tissues based on results of gross pathological examination.

Detailed Clinical Observations and Clinical Observations:
Detailed clinical observations (DCO) were conducted prior to exposure, twice following exposure (test day 1), and daily thereafter. The DCO was conducted on all animals, at approximately the same time each day according to an established format. The
examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (ranked) as outlined in Appendix A. Categorical observations (descriptive) are summarized under the clinical observations
table(s).

Statistics:

Means and standard deviations were calculated for descriptive purposes for chamber concentration (mean only), animal body weights, exposure room temperatures and chamber temperature, humidity, and airflow. No statistical analysis to determine the
LC50 was performed as no mortality was observed.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.8 mg/L air

Exp. duration:

4 h

Mortality:

All animals survived the four-hour exposure to the test material as well as the two-week post-exposure period.

Clinical signs:

other: Clinical effects noted during the four-hour exposure period were limited to soiling of the haircoat in 2/5 male and 3/5 female rats. In-life observations noted post-exposure included perineal soiling in 5/5 female rats and extensive body soiling in 2/5 f

Body weight:

Mean body weight losses of 3.3% and 2.1% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8 and all rats continued to gain weight until the scheduled test
day 15 necropsy.

Gross pathology:

There were no treatment-related visible lesions noted in any of the rats exposed to PRIVENTZ™ Liquid at the test day 15-scheduled necropsy. Non-treatment related visible lesions noted at necropsy were limited to necrotic fat in one
male rat.

Any other information on results incl. tables

Chamber Summary Data:

The resulting time-weighted average concentration was 5.80 mg/L; the nominal concentration was 20.0 mg/L. The differences between the gravimetric and the nominal concentration were due to the loss of test material coating the walls of the generation apparatus and exposure chamber, and the inefficiency of the generation system employed.

The average chamber temperature and relative humidity were 20.2 ± 0.1°C and 69.4 ± 11.6%, respectively. The average exposure room temperature was 21.0 ± 0.1°C. The chamber O2 level was determined to be 20.1% and the CO2 level was determined to be 483ppm. Airflow was maintained at 49 liters per minute.

Based on two determinations, the mean MMAD of the particles was 2.17 microns with an average geometric standard deviation of 2.19 microns. Approximately 21.83% of the particle mass was contained in a size fraction with an aerodynamic diameter less than 1 micron.

Approximately 90.6% of the particulate mass was present in size fractions with an aerodynamic diameter less than 6 microns.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the data, the four-hour LC50 of inhaled PRIVENTZ™ Liquid exceeded the limit test concentration of 5 mg/L and is greater than 5.80 mg/L for male and female F344/DuCrl rats.

Executive summary:

This study was conducted to determine the acute inhalation toxicological properties of PRIVENTZ™ Liquid. Groups of five rats/sex were exposed for four hours, using a nose-only inhalation exposure system, to a time-weighted average chamber concentration of 5.80 mg PRIVENTZ™ Liquid per liter of air. The mass median aerodynamic diameter (MMAD) of the PRIVENTZ™ Liquid aerosol present in the exposure chamber test atmosphere averaged 2.17 microns with an average geometric standard deviation of 2.19 microns.

All animals survived the four-hour exposure to the test material as well as the 14-day postexposure period. Clinical effects noted during the four-hour exposure period were limited to soiling of the haircoat in 2/5 male and 3/5 female rats. In-life observations noted postexposure included perineal soiling in 5/5 female rats and extensive body soiling in 2/5 female rats. All rats appeared normal by test day 5. Mean body weight losses of 3.3 and 2.1% were noted for male and female rats, respectively, on test day 2. Pre-exposure mean body weight values were exceeded on test day 8. There were no visible treatment-related lesions noted in any of the rats exposed to PRIVENTZ™ Liquid at the test day 15-scheduled necropsy. Nontreatment related visible lesions noted at necropsy were limited to necrotic fat in one male rat.

Based on these data, the four-hour LC50 of inhaled, aerosolized PRIVENTZ™ Liquid exceeded the limit test concentration of 5 mg/L and is greater than 5.80 mg/L for male and female F344/DuCrl rats.