Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 5 – 19, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with EU test guidelines in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
The test substance, identified as HISOLVE EDM, Lot #: 3202Z, was received on October 24, 2013 and November 26, 2013 and was further identified with PSL Reference Number 131024-19H and 131126-5H. The test substance was stored at room temperature. The sample was aerosolized as received, was shaken before dispensing and kept on a magnetic stirrer during aerosolization. Documentation of the methods of synthesis, fabrication, or derivation of the test substance is retained by the Sponsor.
The following information related to the characterization of the test substance was provided by the Sponsor:
Composition: Diethylene Glycol Ethyl Methyl Ether – 100%, CAS # 1002-67-1
Physical Description: Colorless liquid
Solubility: Soluble in water.
Stability: Test substance was expected to be stable for the duration of testing.
Expiration Date: June 5, 2014

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals Number of Animals: 10 Sex: 5 Males and 5 Females. Females assigned to test were nulliparous and non-pregnant. Species/Strain: Rat/Sprague-Dawley derived, albino. Age/Body weight: Young adult (10 weeks)/males 309-380 grams and females 223-259 grams at experimental start. Source: Received from Harlan Laboratories, Inc. on November 13, 2013. Husbandry Housing: The animals were singly housed 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 (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week. Animal Room Temperature and Relative Humidity Ranges: 19-23 °C and 43-55%, respectively. Animal Room Air Changes/Hour: 11. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs. Photoperiod: 12-hour light/dark cycle Acclimation Period: 22 daysFood: Harlan Teklad Global 16% Protein Rodent Diet® 2016. The diet was available ad libitum, except during the exposure. Water: Filtered tap water was supplied ad libitum, except during the exposure. Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs. Identification Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal. 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 37800, constituted unique identification.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Pre-Test Trials 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). In these trials, the following adjustments were made in an attempt to achieve these objectives: Air Pressure: constant Compressed Generator Airflow: constant Compressed Mixing Airflow: constant Total Airflow: constant Pump Setting: varied Liquid Pump: constant Tube Size: constant Atomization System: constant Clean-Out Needle and Fluid Cap: constant Air Cap: constant Vacuum Pump: constant Sampling Time: constant The procedures and aerosolization equipment used in the full test were based on the results of pre-test trial number 2 which provided a gravimetric concentration of 5.00 mg/L and a mass median aerodynamic diameter of 2.33 μm. Inhalation Procedures The exposure chamber, air supply and equipment used to measure particle size distribution, airflow and chamber concentration were the same as used during the appropriate pre-test trials and are described below. Nose-Only Exposure Chamber: A nose-only inhalation chamber with an internal volume of approximately 28 liters (Nose-Only Inhalation Chamber, ADG Developments LTD) was used for exposure. Animals were individually housed in polycarbonate holding tubes which seal to the chamber with an “O” ring during exposure. The base unit terminates the chamber with a 0.5-inch diameter tube for discharged air. Air Supply: Approximately 30.0 liters per minute (Lpm) of filtered generator air was supplied by an air compressor (Powerex Model: SES050822) to the spray atomization nozzle. An additional 6.0 Lpm of filtered mixing air from the same air compressor was introduced into the chamber to help uniformly distribute the test atmosphere by creating a vortex at the chamber inlet. Compressed airflow was measured with a Mass Flow Controller (Omega, Model #FMA-5541) for generator air and Mass Flow Meter (Omega, Model #FMA-5613) for mixing air. Chamber airflow was monitored throughout the exposure period and recorded periodically. Total airflow was 36.0 Lpm. Based on the volume of the inhalation chamber, this airflow provided approximately 77 air changes per hour during the study. The exposure was conducted under slightly negative pressure. Ambient Conditions: The exposure chamber temperature and relative humidity ranges during exposure were 20-21 °C and 43-46%, respectively. The room temperature and relative humidity ranges during exposure were 20-21 °C and 49-51%, respectively. The measurements inside the exposure chamber and room conditions were measured with a Temperature-Humidity Monitor (Fisher Scientific, Model #11-661-18). Temperature and relative humidity values were recorded every 15 minutes for the first hour of exposure and approximately every 15 or 30 minutes thereafter. Atmosphere Generation: The test atmosphere was generated using a ¼ inch JCO nebulizer, FC3 clean-out needle and fluid cap and 70 SS air cap (all from Spraying Systems Co.). Compressed generator/mixing air was supplied at 30/30 psi, respectively. The test substance was metered to the atomization nozzle through size 14 PharMed® BPT tubing, using a peristaltic pump (Masterflex®, Model #7520-35). Chamber Concentration Measurements: Gravimetric samples were withdrawn at 6 intervals from the breathing zone of the animals. Samples were collected using 37 mm glass fiber filters (WhatmanTM GF/B) in a filter holder attached by ¼ inch Tygon® tubing to a vacuum pump (GAST, Model #1531-107B-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 1 minute at airflows of 4.0 Lpm. Sample airflows were measured using a Mass Flow Controller (Aalborg, Model #GFC-17).Particle Size Distribution: An eight-stage 1 ACFM Andersen Ambient Particle Sizing Sampler 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 mass median aerodynamic diameter and geometric standard deviation were calculated using two-cycle logarithmic probit axes. Exposure Period: The animals were exposed to the test atmosphere for 4 hours and 4 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 1.79 and 3.58 minutes, respectively. At the end of the exposure period, the generation was terminated and the chamber was operated for a further 15 minutes with clean air. At the end of this period the animals were removed from the exposure tube. Prior to being returned to their cages excess test substance was removed from the fur of each animal.
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
5.0 mg/L
No. of animals per sex per dose:
5 males & 5 females
Control animals:
no
Details on study design:
Selection of Animals On the day of and prior to exposure, the rats were examined for health and weighed. Ten healthy naive rats (five males and five females; not previously tested) were selected for test. Body Weights Individual body weights of the animals were recorded prior to test substance exposure (initial) and again on Days 1, 3, 7 and 14 (termination). Cage-Side Observations All animals were observed for mortality during the exposure period. The animals were examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube 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. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma. Necropsy All rats were euthanized via CO2 inhalation on Day 14. Gross necropsies were performed on all animals. Tissues and organs of the thoracic and abdominal cavities were examined.
Statistics:
Statistical analysis was limited to the calculation of the mean and standard deviation.

Results and discussion

Preliminary study:
Preliminary study not required
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.14 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
All animals survived exposure to the test atmosphere.
Clinical signs:
other: Following exposure, all rats exhibited irregular respiration, with one male exhibiting hypoactivity. However, all animals recovered by Day 4 and appeared active and healthy for the remainder of the 14-day observation period.
Body weight:
Although all animals lost body weight by Day 1, and one female by Day 7, all showed a continued weight gain thereafter through Day 14.
Gross pathology:
No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.
Other findings:
No further findings reported

Any other information on results incl. tables

PRE-TEST EXPOSURE TRIALS

Trial No.

Generator/ Mixing Air Pressure (psi)

Compressed Generator Air (Lpm)

Compressed Mixing Air (Lpm)

Total Air Volume (Lpm)

Pump Setting

Chamber Conc. (mg/L)

Particle Size Sampled

1

30/30

30.0

6.0

36.0

8.0

6.48

No

2

30/30

30.0

6.0

36.0

6.0

5.00

Yes

 

SUMMARY OF PRE-TEST EXPOSURE TRIAL

Trial No.

Chamber Concentration (mg/L)

Mass Median Aerodynamic Diameter (μm)

2

5.00

2.33

 

GRAVIMETRIC CHAMBER CONCENTRATIONS

Sample Number

Time of Sample (hour)

Mass Collected (mg)

Airflow Sampled (Lpm)

Collection Time (min)

Chamber Conc. (mg/L)

1

0.5

22.0

4.0

1

5.50

2

1

20.2

4.0

1

5.05

3

2

20.4

4.0

1

5.10

4

2.5

20.2

4.0

1

5.05

5

3.5

20.3

4.0

1

5.08

6

3.75

20.1

4.0

1

5.03

Average ± Standard Deviation

5.14 ± 0.18

 

NOMINAL CHAMBER CONCENTRATION

Exposure Level (mg/L)

Total Test Substance Used (g)

Average Total Airflow (Lpm)

Total Time of Exposure (min)

Nominal Concentration (mg/L)

5.14

312.3

36.0

244

35.55

 

PARTICLE SIZE DISTRIBUTION

Stage

Effective Cut off Diameter (μm)

% of Total Particles Captured (by weight)

Cumulative (%)1

Sample 1

0

9.0

9.2

90.8

1

5.8

11.8

78.9

2

4.7

13.2

65.8

3

3.3

26.3

39.5

4

2.1

14.5

25.0

5

2.1

7.9

17.1

6

0.7

5.3

11.8

7

0.4

3.9

7.9

F

0.0

7.9

0.0

Sample 2

0

9.0

7.0

93.0

1

5.8

8.8

84.2

2

4.7

7.0

77.2

3

3.3

17.5

59.6

4

2.1

28.1

31.6

5

1.1

7.0

24.6

6

0.7

7.0

17.5

7

0.4

7.0

10.5

F

0.0

10.5

0.0

1Percent of particles smaller than corresponding effective cut off diameter

 

SUMMARY OF PARTICLE SIZE DISTRIBUTION

Sample No.

Time of Sample (hour)

Collection Time (minutes)

Mass Median Aerodynamic Diameter (μm)

Geometric Standard Deviation

1

1.5

1

2.94

2.78

2

3

1

2.29

2.65

Mean

2.62

2.72

 

INDIVIDUAL BODY WEIGHTS

Animal No.

Sex

Body Weight (g)

Initial

Day 1

Day 3

Day 7

Day 14

3301

M

330

352

376

392

443

3302

M

380

369

390

407

453

3303

M

374

353

366

375

414

3304

M

309

292

304

320

355

3305

M

336

313

335

345

390

3306

F

226

216

227

226

249

3307

F

223

214

219

223

250

3308

F

237

234

238

241

265

3309

F

251

242

251

258

286

3310

F

259

245

259

255

283

 

 

INDIVIDUAL CAGE-SIDE OBSERVATIONS

Animal number

Findings

Day of Occurrence

MALES

3301, 3303 – 3305

Irregular respiration

Active and healthy

CR1-2

3-14

3302

Irregular respiration

Hypoactivity

Active and healthy

CR-3

1-2

4-14

FEMALES

3306, 3307, 3309

Irregular respiration

Active and healthy

CR-3

4-14

3308, 3310

Irregular respiration

Active and healthy

CR-2

3-14

1CR – removal from the exposure tube

 

INDIVIDUAL NECROPSY OBSERVATIONS

Animal Number

Tissue

Findings

MALES

3301 – 3305

All tissues and organs

No gross abnormalities

FEMALES

3306 – 3310

All tissues and organs

No gross abnormalities

 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the single exposure acute inhalation LC50 of HISOLVE EDM is greater than 5.14 mg/L in male and female rats.
Executive summary:

This study was conducted at Product Safety Labs’ (PSL) test facility at 2394 US Highway 130, Dayton, New Jersey 08810. The Study Director for this study was Daniel Merrill, BS, MBA. The primary scientist for this study was Mark Schooley, with contributions by Jessica Beyenhof, BS, Harry Maselli, ALAT and Matthew Notta, BS. This study was conducted to comply with the Good Laboratory Practice (GLP) regulations as defined in:

-EC Directive 2004/10/EC, Official Journal of the European Union, L50/44, Feb. 20, 2004

and based on the following testing guideline:

-Official Journal of the European Communities. Methods for the Determination of Toxicity and Other Health Effects, Part B.2 (Acute Toxicity Inhalation) Commission Regulation (EC) No. 440/2008

 

An acute inhalation toxicity test was conducted with rats to determine the potential for HISOLVE EDM to produce toxicity from a single exposure via the inhalation (nose-only exposure) route. Under the conditions of this study, the single exposure acute inhalation LC50 of the test substance is greater than 5.14 mg/L in male and female rats.

After establishing the desired generation procedures during 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 substance 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 14 days following exposure. Body weights were recorded prior to exposure and again on Days 1, 3, 7 and 14 (termination). Necropsies were performed on all animals at terminal sacrifice.

The gravimetric chamber concentration was 5.14 mg/L. The mass median aerodynamic diameter was calculated to be 2.62 μm based on graphic analysis of the particle size distribution as measured with 1 ACFM Andersen Ambient Particle Sizing Sampler.

All animals survived exposure to the test atmosphere. Following exposure, all rats exhibited irregular respiration, with one male exhibiting hypoactivity. However, all animals recovered by Day 4 and appeared active and healthy for the remainder of the 14-day observation period. Although all animals lost body weight by Day 1, and one female by Day 7, all showed a continued weight gain thereafter through Day 14. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.