Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant OECD guideline study, available as unpublished report, only 2-week study but furthermore no restrictions, adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Principles of method if other than guideline:
The study is conducted similarly to OECD testing guideline 403, but repeatedly during 2 weeks
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3G8 / Triethylene Glycol Di-2-Ethylhexanoate
- Physical state: straw colored liquid
- Purity: 98.3%
- Impurities (identity and concentrations): not provided
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 6 weeks old on the day of arrival
- Housing: Except during exposure, animals were housed singly in stainless steel wire-mesh cages
- Diet (e.g. ad libitum): ad libitum except during exposure (PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002)
- Water (e.g. ad libitum): ad libitum except during exposure (tap water)
- Acclimation period: Rats were quarantined after arrival for 3 days prior to testing, further temporarily identified by cage identification, weighed and observed for signs of disease or injury. Then rats were assigned to groups by a computerized stratified randomization program. At study start, the animals were 8 weeks old and weighed between 241 and 286 grams.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Relative humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle
IN-LIFE DATES: From: January 18, 2005 To: February 21, 2005

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Mass median aerodynamic diameter (MMAD) and other conditions: See Table 2
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus and exposure chamber volume: The exposure chamber was constructed of glass (cylindrical) with a nominal internal volume of 34 L. A polycarbonate baffle inside the chamber promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: During exposure, animals were individually restrained in perforated stainless steel cylinders with conical nose pieces. The restrainers were inserted into a polymethylmethacrylate faceplate attached to the exposure chamber so that the nose of each animal extended into the exposure chamber.
- System of generating particulates/aerosols: Chamber atmospheres were generated by aerosolization of the test substance in air with a Spraying Systems nebulizer. The test substance was metered into the nebulizer with a Harvard Apparatus model 22 syringe infusion pump. Filtered, high-pressure air, metered into the nebulizer by a Brooks model 0154E mass flow controller, carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were controlled by varying the rate of the infusion pump.
- Treatment of exhaust air: Test atmospheres were exhausted through a dry-ice cold trap followed by an MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood.
- Temperature, humidity, pressure in air chamber: Chamber temperature was 24°C, relative humidity ranged from 46 - 50%, airflow was 16.0 L/min and the oxygen concentration was 21.0%.

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure the atmospheric concentration of the test substance was determined by gravimetric analysis at approximately 30-minute intervals in the test chamber. Known volumes of chamber atmosphere were drawn from the sampling port through a 25 mm filter cassette containing a pre-weighed glass fiber (Type A/E) filter. The filters were weighed on a Cahn model C-31 Microbalance®. The atmospheric concentration of the test substance was calculated from the difference between the pre- and postsampling filter weights divided by the volume of chamber atmosphere sampled.- Particle size distribution: MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The atmosphere generated in this study was considered to be respirable in rats, the mass median aerodynamic diameter was 2.0 μm ± 2.0 (MMAD ± GSD), with 97% of the aerosol being less than 10 μm MMAD.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample to determine aerosol size distribution (mass median aerodynamic diameter and percent
aerosol less than 1,3, and 10 µm diameter) was taken fiom each test chamber for Groups III, V, and VII weekly with a Sierra senes 210 cyclone preseparatortcascade impactor and sierram series 110 constant flow air sampler.
Duration of treatment / exposure:
Each group of rats was exposed 6 hours/day. The starting time of each exposure was defined as the time when the generation system was turned on. The ending time of each exposure was defined as the time when the generation system is turned off. Rats were exposed to the test substance during both the time it took for the chamber to reach concentration, and the time it took for the test substance to be purged from the chamber. After each exposure, rats were returned to their cages. The exposure period was defined as the period between initiation of the first exposure and completion of the last exposure.
Frequency of treatment:
Each group of rats was over a 2-week period (weekends and holidays excluded) for a total of 9 exposures.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 10 ± 0.35, 300 ± 5.8, 1000 ± 19 mg/m³ (Groups I, III, V, VII, respectively)
Basis:

No. of animals per sex per dose:
10 male animals/group
Control animals:
yes
Details on study design:
At the conclusion of the 2-week exposure period, 5 rats per group were allowed to recover for 2 weeks. During the recovery period observations were continued.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time scheudule: daily during 14 days

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: individual examination immediately following exposure on the days of dosing.

BODY WEIGHT: yes
- Time scheudule: twice weekly – all animals

FOOD CONSUMPTION: no
FOOD EFFICIENCY: no
WATER CONSUMPTION: no
OPHTHALMOSCOPIC EXAMINATION: no
HAEMATOLOGY: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Anaesthetic: carbon dioxide anesthesia
- Animals fasted: yes (15h)
- No. of animals: all animals on day 11; 5 animals/group on day 25
- Parameters: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Animals fasted: yes (15h)
- No. of animals: all animals on day 11; 5 animals/group on day 25
- Parameters: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic phosphate, sodium, potassium, chloride
URINALYSIS: yes
- Time schedule: day 11 (1 day post-exposure); day 25 (2 weeks recovery)
- Metabolism cages used for collection: yes (15h)
- Animals fasted: yes (15h)
- Parameters: quality, color, clarity, ketone, bilirubin, blood, volume, osmolality, pH, glucose, urobilinogen, protein, microscopic urine sediment examination
NEUROBEHAVIOURAL EXAMINATION: no

Sacrifice and pathology:
- Necropsy (final body weights, organ weights, macroscopic examination): 5 animals/group on day 11 (1 day post-exposure) ; 5 animals/group on day 25 (2 weeks recovery)
- Group organ weight means and ratios (% body weight and % brain weight) were calculated.

HISTOPATHOLOGY: yes
- all organs were processed and examined.
Statistics:
See Table 1.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
other: LD50
Effect level:
> 2 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

TABLE STILL TO BE ADDED APPENDIXES REQUIRED

Applicant's summary and conclusion

Conclusions:
Based upon the observations, the NOAEL = 1000 mg/m3
Executive summary:

Four groups of 10 male CRI:CD(SD)IGS BR rats were exposed to aerosol atmospheres (1.9-2.4 µm) of 3G0 at 0 (control), 10+0.35,300+5.8 or 1000+19 mg/m3(mean+S.E.M.). Animals were exposed nose only, 6 hours a day for a total of 9 exposures over a 2-week period. The dosing period was followed by a recovery period in half of the animals No test substance related effects were observed in clinical observations, body weights, clinical pathology, gross necropsy observations and microscopic pathology.Based upon the observation of no significant effects following exposure to 3G0, the no observed- Adverse effect level (NOAEL) for a 2-week repeated inhalation exposure was 1000 mg/m3for male rats.